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Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN.

Menck K, Scharf C, Bleckmann A, Dyck L, Rost U, Wenzel D, Dhople VM, Siam L, Pukrop T, Binder C, Klemm F - J Mol Cell Biol (2014)

Bottom Line: Uptake of T-MV is essential for the proinvasive effect.Interestingly, the effect of EMMPRIN-bearing MV is not mediated by matrix metalloproteinases but by activation of the p38/MAPK signaling pathway in the tumor cells.In conclusion, T-MV stimulate cancer cell invasion via a direct feedback mechanism dependent on highly glycosylated EMMPRIN.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology/Oncology, University Medical Center Göttingen, 37099 Göttingen, Germany.

No MeSH data available.


Related in: MedlinePlus

MV uptake is important for their proinvasive function. (A) Fluorescence microscopy showing the uptake of MV (5 µg/ml) labeled with the red fluorescent dye PKH26 by MCF-7 cells after 24 h of stimulation (magnification, 10×; inserts: bright field). (B) Uptake of PKH26-labeled T-MV (5 µg/ml) into MCF-7 cells preincubated with the endocytosis inhibitor dynasore (Dyn) was quantified by flow cytometry (mean ± SD, n = 4, *P < 0.01, **P < 0.001; MFI, mean fluorescence intensity). (C) Microinvasion assay of MCF-7 cells pretreated with Dyn (12.5 µM) prior to stimulation with T-MV (mean ± SD, n = 3, *P < 0.001). (D) Microinvasion assay of MCF-7 cells exposed to heat-inactivated (5 min, 95°C) T-MV (mean ± SD, n = 3, *P < 0.001).
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MJU047F2: MV uptake is important for their proinvasive function. (A) Fluorescence microscopy showing the uptake of MV (5 µg/ml) labeled with the red fluorescent dye PKH26 by MCF-7 cells after 24 h of stimulation (magnification, 10×; inserts: bright field). (B) Uptake of PKH26-labeled T-MV (5 µg/ml) into MCF-7 cells preincubated with the endocytosis inhibitor dynasore (Dyn) was quantified by flow cytometry (mean ± SD, n = 4, *P < 0.01, **P < 0.001; MFI, mean fluorescence intensity). (C) Microinvasion assay of MCF-7 cells pretreated with Dyn (12.5 µM) prior to stimulation with T-MV (mean ± SD, n = 3, *P < 0.001). (D) Microinvasion assay of MCF-7 cells exposed to heat-inactivated (5 min, 95°C) T-MV (mean ± SD, n = 3, *P < 0.001).

Mentions: We next asked whether MV are incorporated by tumor cells and whether this differs between MV populations explaining their differential features. Time course experiments revealed that a major part of T-MVM was taken up into MCF-7 cells after 24 h of stimulation (Supplementary Figure S2A). However, all of the investigated MV populations, regardless of their origin, were incorporated into both breast cancer cell lines at this time point (Figure 2A, Supplementary Figure S2B and C). Although the uptake of heterologous MV, including proinvasive T-MVS and non-proinvasive hTERT-MV, into MCF-7 cells seemed to be lower compared with the uptake of autologous T-MVM, the tumor cells still ingested a significant portion of all MV populations. This suggests that MV uptake per se does not differ between proinvasive and non-proinvasive MV. To investigate whether MV incorporation is nonetheless essential for MV function, we tried to antagonize T-MV uptake into breast cancer cells with known inhibitors of endocytosis at concentrations that did not interfere with cell viability (Supplementary Figure S2D and E). Neither inhibition of clathrin-mediated endocytosis with dansylcadaverine, nor interfering with caveolae-dependent endocytosis by cholesterol depletion in the recipient cells with filipin III decreased T-MV ingestion (Supplementary Figure S2F). Thus, T-MV incorporation occurs independent of either clathrin- or caveolin-mediated endocytosis. However, pharmacological inhibition of dynamin with dynasore led to a significant concentration-dependent reduction of T-MV uptake into MCF-7 (Figure 2B) as well as SK-BR-3 (Supplementary Figure S2F). Consistently, T-MV-induced invasion was significantly decreased after dynasore treatment (Figure 2C, Supplementary Figure S2G). Taken together, these data imply a dynamin-dependent uptake of T-MV as an important requirement for MV-triggered invasion. Furthermore, heating of T-MV significantly decreased their proinvasive phenotype (Figure 2D), indicating that their tumor-promoting effect is not mediated by transferring lipids, but by proteins most likely differentially expressed on T-MV and benign MV.Figure 2


Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN.

Menck K, Scharf C, Bleckmann A, Dyck L, Rost U, Wenzel D, Dhople VM, Siam L, Pukrop T, Binder C, Klemm F - J Mol Cell Biol (2014)

MV uptake is important for their proinvasive function. (A) Fluorescence microscopy showing the uptake of MV (5 µg/ml) labeled with the red fluorescent dye PKH26 by MCF-7 cells after 24 h of stimulation (magnification, 10×; inserts: bright field). (B) Uptake of PKH26-labeled T-MV (5 µg/ml) into MCF-7 cells preincubated with the endocytosis inhibitor dynasore (Dyn) was quantified by flow cytometry (mean ± SD, n = 4, *P < 0.01, **P < 0.001; MFI, mean fluorescence intensity). (C) Microinvasion assay of MCF-7 cells pretreated with Dyn (12.5 µM) prior to stimulation with T-MV (mean ± SD, n = 3, *P < 0.001). (D) Microinvasion assay of MCF-7 cells exposed to heat-inactivated (5 min, 95°C) T-MV (mean ± SD, n = 3, *P < 0.001).
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Related In: Results  -  Collection

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MJU047F2: MV uptake is important for their proinvasive function. (A) Fluorescence microscopy showing the uptake of MV (5 µg/ml) labeled with the red fluorescent dye PKH26 by MCF-7 cells after 24 h of stimulation (magnification, 10×; inserts: bright field). (B) Uptake of PKH26-labeled T-MV (5 µg/ml) into MCF-7 cells preincubated with the endocytosis inhibitor dynasore (Dyn) was quantified by flow cytometry (mean ± SD, n = 4, *P < 0.01, **P < 0.001; MFI, mean fluorescence intensity). (C) Microinvasion assay of MCF-7 cells pretreated with Dyn (12.5 µM) prior to stimulation with T-MV (mean ± SD, n = 3, *P < 0.001). (D) Microinvasion assay of MCF-7 cells exposed to heat-inactivated (5 min, 95°C) T-MV (mean ± SD, n = 3, *P < 0.001).
Mentions: We next asked whether MV are incorporated by tumor cells and whether this differs between MV populations explaining their differential features. Time course experiments revealed that a major part of T-MVM was taken up into MCF-7 cells after 24 h of stimulation (Supplementary Figure S2A). However, all of the investigated MV populations, regardless of their origin, were incorporated into both breast cancer cell lines at this time point (Figure 2A, Supplementary Figure S2B and C). Although the uptake of heterologous MV, including proinvasive T-MVS and non-proinvasive hTERT-MV, into MCF-7 cells seemed to be lower compared with the uptake of autologous T-MVM, the tumor cells still ingested a significant portion of all MV populations. This suggests that MV uptake per se does not differ between proinvasive and non-proinvasive MV. To investigate whether MV incorporation is nonetheless essential for MV function, we tried to antagonize T-MV uptake into breast cancer cells with known inhibitors of endocytosis at concentrations that did not interfere with cell viability (Supplementary Figure S2D and E). Neither inhibition of clathrin-mediated endocytosis with dansylcadaverine, nor interfering with caveolae-dependent endocytosis by cholesterol depletion in the recipient cells with filipin III decreased T-MV ingestion (Supplementary Figure S2F). Thus, T-MV incorporation occurs independent of either clathrin- or caveolin-mediated endocytosis. However, pharmacological inhibition of dynamin with dynasore led to a significant concentration-dependent reduction of T-MV uptake into MCF-7 (Figure 2B) as well as SK-BR-3 (Supplementary Figure S2F). Consistently, T-MV-induced invasion was significantly decreased after dynasore treatment (Figure 2C, Supplementary Figure S2G). Taken together, these data imply a dynamin-dependent uptake of T-MV as an important requirement for MV-triggered invasion. Furthermore, heating of T-MV significantly decreased their proinvasive phenotype (Figure 2D), indicating that their tumor-promoting effect is not mediated by transferring lipids, but by proteins most likely differentially expressed on T-MV and benign MV.Figure 2

Bottom Line: Uptake of T-MV is essential for the proinvasive effect.Interestingly, the effect of EMMPRIN-bearing MV is not mediated by matrix metalloproteinases but by activation of the p38/MAPK signaling pathway in the tumor cells.In conclusion, T-MV stimulate cancer cell invasion via a direct feedback mechanism dependent on highly glycosylated EMMPRIN.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology/Oncology, University Medical Center Göttingen, 37099 Göttingen, Germany.

No MeSH data available.


Related in: MedlinePlus