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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

HG-EMMPRIN on T-MV is glycosylated at N160 and N268. (A) Expression of EMMPRIN (EMP) isoforms EMP-2, EMP-3, and EMP-4 in human breast (cancer) cells and in brain metastases from eight breast cancer patients (BC brain mets) (qRT–PCR, mean, n = 3). EMP-3 and EMP-4 were not detectable (n.d.) in hTERT-HME1 cells. (B) Schematic representation of EMP-2 with the three predicted N-glycosylation sites. (C) Mass spectrometry of the three predicted EMMPRIN N-glycosylation sites (see text for further details). (D) Microinvasion assay of MCF-7 cells preincubated with specific blocking peptides against the two EMMPRIN glycosylation sites (P-N160, P-N268) and stimulated with T-MV (mean ± SD, n = 3, *P < 0.05, **P < 0.01). Random control peptides (P-N160rd, P-N268rd) were used as controls.
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MJU047F5: HG-EMMPRIN on T-MV is glycosylated at N160 and N268. (A) Expression of EMMPRIN (EMP) isoforms EMP-2, EMP-3, and EMP-4 in human breast (cancer) cells and in brain metastases from eight breast cancer patients (BC brain mets) (qRT–PCR, mean, n = 3). EMP-3 and EMP-4 were not detectable (n.d.) in hTERT-HME1 cells. (B) Schematic representation of EMP-2 with the three predicted N-glycosylation sites. (C) Mass spectrometry of the three predicted EMMPRIN N-glycosylation sites (see text for further details). (D) Microinvasion assay of MCF-7 cells preincubated with specific blocking peptides against the two EMMPRIN glycosylation sites (P-N160, P-N268) and stimulated with T-MV (mean ± SD, n = 3, *P < 0.05, **P < 0.01). Random control peptides (P-N160rd, P-N268rd) were used as controls.

Mentions: To further analyze EMMPRIN glycosylation patterns on T-MV, T-MVM were digested with PNGaseF and all EMMPRIN bands detected by western blotting (see Figure 4B) were excised from one-dimensional SDS electrophoresis gels and subjected to LC-MS/MS. Alignment of the assigned peptides displayed the highest coverage for the isoforms EMMPRIN-2 and EMMPRIN-3, whereas peptides specific for EMMPRIN-1 and EMMPRIN-4 could not be identified (Supplementary Figure S5A). Spectral counting revealed that the N-terminal fragment of EMMPRIN-2 was present in a ratio of 10:1 compared with EMMPRIN-3. This is in line with an mRNA screening of three cell lines and the human brain metastases from eight patients with primary breast cancer, which showed that EMMPRIN-2 is expressed at very high levels in vitro and in vivo compared with the other two isoforms, EMMPRIN-3 and EMMPRIN-4 (Figure 5A). These results suggest that EMMPRIN-2 is the predominant isoform on T-MV. However, since it is also present on hTERT-MV, its expression per se is not sufficient for the proinvasive MV effect, which requires additional posttranslational modifications, in particular strong glycosylation.Figure 5


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)

HG-EMMPRIN on T-MV is glycosylated at N160 and N268. (A) Expression of EMMPRIN (EMP) isoforms EMP-2, EMP-3, and EMP-4 in human breast (cancer) cells and in brain metastases from eight breast cancer patients (BC brain mets) (qRT–PCR, mean, n = 3). EMP-3 and EMP-4 were not detectable (n.d.) in hTERT-HME1 cells. (B) Schematic representation of EMP-2 with the three predicted N-glycosylation sites. (C) Mass spectrometry of the three predicted EMMPRIN N-glycosylation sites (see text for further details). (D) Microinvasion assay of MCF-7 cells preincubated with specific blocking peptides against the two EMMPRIN glycosylation sites (P-N160, P-N268) and stimulated with T-MV (mean ± SD, n = 3, *P < 0.05, **P < 0.01). Random control peptides (P-N160rd, P-N268rd) were used as controls.
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MJU047F5: HG-EMMPRIN on T-MV is glycosylated at N160 and N268. (A) Expression of EMMPRIN (EMP) isoforms EMP-2, EMP-3, and EMP-4 in human breast (cancer) cells and in brain metastases from eight breast cancer patients (BC brain mets) (qRT–PCR, mean, n = 3). EMP-3 and EMP-4 were not detectable (n.d.) in hTERT-HME1 cells. (B) Schematic representation of EMP-2 with the three predicted N-glycosylation sites. (C) Mass spectrometry of the three predicted EMMPRIN N-glycosylation sites (see text for further details). (D) Microinvasion assay of MCF-7 cells preincubated with specific blocking peptides against the two EMMPRIN glycosylation sites (P-N160, P-N268) and stimulated with T-MV (mean ± SD, n = 3, *P < 0.05, **P < 0.01). Random control peptides (P-N160rd, P-N268rd) were used as controls.
Mentions: To further analyze EMMPRIN glycosylation patterns on T-MV, T-MVM were digested with PNGaseF and all EMMPRIN bands detected by western blotting (see Figure 4B) were excised from one-dimensional SDS electrophoresis gels and subjected to LC-MS/MS. Alignment of the assigned peptides displayed the highest coverage for the isoforms EMMPRIN-2 and EMMPRIN-3, whereas peptides specific for EMMPRIN-1 and EMMPRIN-4 could not be identified (Supplementary Figure S5A). Spectral counting revealed that the N-terminal fragment of EMMPRIN-2 was present in a ratio of 10:1 compared with EMMPRIN-3. This is in line with an mRNA screening of three cell lines and the human brain metastases from eight patients with primary breast cancer, which showed that EMMPRIN-2 is expressed at very high levels in vitro and in vivo compared with the other two isoforms, EMMPRIN-3 and EMMPRIN-4 (Figure 5A). These results suggest that EMMPRIN-2 is the predominant isoform on T-MV. However, since it is also present on hTERT-MV, its expression per se is not sufficient for the proinvasive MV effect, which requires additional posttranslational modifications, in particular strong glycosylation.Figure 5

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