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Periostin suppression induces decorin secretion leading to reduced breast cancer cell motility and invasion.

Ishiba T, Nagahara M, Nakagawa T, Sato T, Ishikawa T, Uetake H, Sugihara K, Miki Y, Nakanishi A - Sci Rep (2014)

Bottom Line: The ability of cancer cells to metastasize is dependent on the interactions between their cell-surface molecules and the microenvironment.However, the tumor microenvironment, especially the cancer-associated stroma, is poorly understood.Our results reveal the molecular details of the periostin-decorin complex in both phyllodes tumor tissues and breast cancer cells; this interaction may represent a novel target for anti-cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University (TMDU) [2] Department of Surgical Oncology, Tokyo Medical and Dental University (TMDU).

ABSTRACT
The ability of cancer cells to metastasize is dependent on the interactions between their cell-surface molecules and the microenvironment. However, the tumor microenvironment, especially the cancer-associated stroma, is poorly understood. To identify proteins present in the stroma, we focused on phyllodes tumors, rare breast tumors that contain breast stromal cells. We compared the expression of proteins between phyllodes tumor and normal tissues using an iTRAQ-based quantitative proteomic approach. Decorin was expressed at reduced levels in phyllodes tumor tissues, whereas periostin was upregulated; this result was validated by immunohistochemical analysis of phyllodes tumors from 35 patients. Additionally, by immunoprecipitation and mass spectrometry, we confirmed that decorin forms a complex with periostin in both phyllodes tumors and BT-20 breast cancer cells. Following siRNA-mediated knockdown of periostin in T-47D cells, secreted decorin in the culture medium could be detected by multiple reaction monitoring (MRM). Furthermore, periostin knockdown in BT-20 cells and overexpression of decorin in MDA-MB-231 cells inhibited cell motility and invasion. Our results reveal the molecular details of the periostin-decorin complex in both phyllodes tumor tissues and breast cancer cells; this interaction may represent a novel target for anti-cancer therapy.

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Related in: MedlinePlus

The detection of secreted decorin in the culture medium using multiple reaction monitoring (MRM).(a) MRM chromatograms of VSPGAFTPLVK fragments and their standard (AQUA: red line) analogues. The peptides in culture medium from siRNA-periostin–treated cells (blue line) or siRNA-control–treated cells (green line) were analyzed using the MRM method. The doubly charged precursor mass was chosen as the Q1 mass, and the y8 fragment ion was chosen as the Q3 mass. Insets contain magnified views. (b) MRM chromatograms for VSPGAFTPLVK fragments and their standard (AQUA) analogues. MRM transitions for the endogenous (blue line) and standard (red line) peptides were monitored. For VSPGAFTPLVK, the doubly charged precursor mass was chosen as the Q1 mass, and the y8 fragment ions were chosen as Q3 mass. Insets contain magnified views. (c) MRM-triggered MS/MS product ion spectra obtained by nanoflow LC/MS/MS, comparing normal tissue digested with trypsin (upper) with decorin secreted from decorin-overexpressing MDA-MB-231 cells (lower). The spectrum of the peptide clearly shows y-ion fragments. (d) Calculated concentration of decorin in cell-culture medium of siRNA-periostin–treated T47D cells and decorin-overexpressing MDA-MB-231 cells (n = 3).
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f4: The detection of secreted decorin in the culture medium using multiple reaction monitoring (MRM).(a) MRM chromatograms of VSPGAFTPLVK fragments and their standard (AQUA: red line) analogues. The peptides in culture medium from siRNA-periostin–treated cells (blue line) or siRNA-control–treated cells (green line) were analyzed using the MRM method. The doubly charged precursor mass was chosen as the Q1 mass, and the y8 fragment ion was chosen as the Q3 mass. Insets contain magnified views. (b) MRM chromatograms for VSPGAFTPLVK fragments and their standard (AQUA) analogues. MRM transitions for the endogenous (blue line) and standard (red line) peptides were monitored. For VSPGAFTPLVK, the doubly charged precursor mass was chosen as the Q1 mass, and the y8 fragment ions were chosen as Q3 mass. Insets contain magnified views. (c) MRM-triggered MS/MS product ion spectra obtained by nanoflow LC/MS/MS, comparing normal tissue digested with trypsin (upper) with decorin secreted from decorin-overexpressing MDA-MB-231 cells (lower). The spectrum of the peptide clearly shows y-ion fragments. (d) Calculated concentration of decorin in cell-culture medium of siRNA-periostin–treated T47D cells and decorin-overexpressing MDA-MB-231 cells (n = 3).

Mentions: We next sought to investigate the functional significance of the interaction between decorin and periostin, both of which are secreted proteins. First, we confirmed that both proteins were expressed in the breast cancer cell lines BT-20 and T-47D; in other cell lines we examined (MCF7, MDA-MB231, and HeLa S3), periostin was present but decorin was not (Figure 3a and Supplementary Figure S2). By co-immunoprecipitation of these proteins from BT-20 lysates, we confirmed that endogenous decorin and periostin interacted in these cells, either directly or indirectly (Figure 3b and Supplementary Figure S3). Immunofluorescence confocal microscopy revealed that decorin and periostin colocalized in BT-20 cells (Figure 3c). Decorin and periostin are components of the extracellular matrix45. We hypothesized that decorin is secreted into the culture medium following treatment with siRNA-periostin. To test this hypothesis, we analyzed secreted decorin in the culture medium by immunoprecipitation, followed by immunoblotting using an anti-decorin antibody, but this antibody was highly cross-reactive and yielded many nonspecific bands(Figure 3d). To overcome this technical obstacle, we used multiple reaction monitoring (MRM) mass spectrometry. In this assay, 5 fmol of standard peptides [VSPGAFTPLVK (13C6, 15N2) or DLPPDTTLLDLQNNK (13C6, 15N2)] was separated by nano-LC, and the MRM transitions were monitored. The peptides were delivered in 5% (v/v) acetonitrile at a concentration of 5 pmol/μl. Figure 4a and Supplementary Figure S4 show an MRM transition for the co-eluting standard and endogenous peptides (VSPGAFTPLVK and DLPPDTTLLDLQNNK from decorin). In medium in which siRNA-periostin–treated cells were cultured, the spectrum peak corresponding to endogenous peptides overlapped with that of the standard peptide. However, in medium from siRNA-control–treated cells, there was no spectrum peak corresponding to endogenous peptides. On the other hand, periostin was detected by immunoblot analysis with anti-periostin antibody in medium from cells treated with siRNA-decorin cells, but not detected in medium from cells treated with siRNA-control or in non-transfected cells (Figure 3e). Next, we investigated whether MDA-MB-231 cells secrete decorin following decorin transfection, because these cells do not normally express decorin (Figure 3a). Figure 4b and Supplementary Figure S5 show two MRM transitions for the co-eluting standard and endogenous peptide (VSPGAFTPLVK from decorin). The decorin peptide sequence VSPGAFTPLVK was detected using nano-LC-MS/MS, and was determined at a 95% confidence level. We confirmed that the MS/MS spectrum of the peptide derived from secreted decorin was consistent with the decorin spectrum determined in normal tissue. This MRM-based assay demonstrated the high accuracy of target detection by MS/MS analysis (Figure 4c). The absolute quantitations are shown in Figure 4d. We calculated the concentration of decorin in cell-culture medium of siRNA-periostin–treated T47D cells and decorin-overexpressing MDA-MB-231 cells (n = 3). The levels of decorin in medium from siRNA-periostin–treated T47D cells and decorin-overexpressing MDA-MB-231 cells were 0.026 ± 0.007 nM and 0.699 ± 0.143 nM, respectively. The peaks of the endogenous peptide in each control were weak and non-detectable (ND).


Periostin suppression induces decorin secretion leading to reduced breast cancer cell motility and invasion.

Ishiba T, Nagahara M, Nakagawa T, Sato T, Ishikawa T, Uetake H, Sugihara K, Miki Y, Nakanishi A - Sci Rep (2014)

The detection of secreted decorin in the culture medium using multiple reaction monitoring (MRM).(a) MRM chromatograms of VSPGAFTPLVK fragments and their standard (AQUA: red line) analogues. The peptides in culture medium from siRNA-periostin–treated cells (blue line) or siRNA-control–treated cells (green line) were analyzed using the MRM method. The doubly charged precursor mass was chosen as the Q1 mass, and the y8 fragment ion was chosen as the Q3 mass. Insets contain magnified views. (b) MRM chromatograms for VSPGAFTPLVK fragments and their standard (AQUA) analogues. MRM transitions for the endogenous (blue line) and standard (red line) peptides were monitored. For VSPGAFTPLVK, the doubly charged precursor mass was chosen as the Q1 mass, and the y8 fragment ions were chosen as Q3 mass. Insets contain magnified views. (c) MRM-triggered MS/MS product ion spectra obtained by nanoflow LC/MS/MS, comparing normal tissue digested with trypsin (upper) with decorin secreted from decorin-overexpressing MDA-MB-231 cells (lower). The spectrum of the peptide clearly shows y-ion fragments. (d) Calculated concentration of decorin in cell-culture medium of siRNA-periostin–treated T47D cells and decorin-overexpressing MDA-MB-231 cells (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4233340&req=5

f4: The detection of secreted decorin in the culture medium using multiple reaction monitoring (MRM).(a) MRM chromatograms of VSPGAFTPLVK fragments and their standard (AQUA: red line) analogues. The peptides in culture medium from siRNA-periostin–treated cells (blue line) or siRNA-control–treated cells (green line) were analyzed using the MRM method. The doubly charged precursor mass was chosen as the Q1 mass, and the y8 fragment ion was chosen as the Q3 mass. Insets contain magnified views. (b) MRM chromatograms for VSPGAFTPLVK fragments and their standard (AQUA) analogues. MRM transitions for the endogenous (blue line) and standard (red line) peptides were monitored. For VSPGAFTPLVK, the doubly charged precursor mass was chosen as the Q1 mass, and the y8 fragment ions were chosen as Q3 mass. Insets contain magnified views. (c) MRM-triggered MS/MS product ion spectra obtained by nanoflow LC/MS/MS, comparing normal tissue digested with trypsin (upper) with decorin secreted from decorin-overexpressing MDA-MB-231 cells (lower). The spectrum of the peptide clearly shows y-ion fragments. (d) Calculated concentration of decorin in cell-culture medium of siRNA-periostin–treated T47D cells and decorin-overexpressing MDA-MB-231 cells (n = 3).
Mentions: We next sought to investigate the functional significance of the interaction between decorin and periostin, both of which are secreted proteins. First, we confirmed that both proteins were expressed in the breast cancer cell lines BT-20 and T-47D; in other cell lines we examined (MCF7, MDA-MB231, and HeLa S3), periostin was present but decorin was not (Figure 3a and Supplementary Figure S2). By co-immunoprecipitation of these proteins from BT-20 lysates, we confirmed that endogenous decorin and periostin interacted in these cells, either directly or indirectly (Figure 3b and Supplementary Figure S3). Immunofluorescence confocal microscopy revealed that decorin and periostin colocalized in BT-20 cells (Figure 3c). Decorin and periostin are components of the extracellular matrix45. We hypothesized that decorin is secreted into the culture medium following treatment with siRNA-periostin. To test this hypothesis, we analyzed secreted decorin in the culture medium by immunoprecipitation, followed by immunoblotting using an anti-decorin antibody, but this antibody was highly cross-reactive and yielded many nonspecific bands(Figure 3d). To overcome this technical obstacle, we used multiple reaction monitoring (MRM) mass spectrometry. In this assay, 5 fmol of standard peptides [VSPGAFTPLVK (13C6, 15N2) or DLPPDTTLLDLQNNK (13C6, 15N2)] was separated by nano-LC, and the MRM transitions were monitored. The peptides were delivered in 5% (v/v) acetonitrile at a concentration of 5 pmol/μl. Figure 4a and Supplementary Figure S4 show an MRM transition for the co-eluting standard and endogenous peptides (VSPGAFTPLVK and DLPPDTTLLDLQNNK from decorin). In medium in which siRNA-periostin–treated cells were cultured, the spectrum peak corresponding to endogenous peptides overlapped with that of the standard peptide. However, in medium from siRNA-control–treated cells, there was no spectrum peak corresponding to endogenous peptides. On the other hand, periostin was detected by immunoblot analysis with anti-periostin antibody in medium from cells treated with siRNA-decorin cells, but not detected in medium from cells treated with siRNA-control or in non-transfected cells (Figure 3e). Next, we investigated whether MDA-MB-231 cells secrete decorin following decorin transfection, because these cells do not normally express decorin (Figure 3a). Figure 4b and Supplementary Figure S5 show two MRM transitions for the co-eluting standard and endogenous peptide (VSPGAFTPLVK from decorin). The decorin peptide sequence VSPGAFTPLVK was detected using nano-LC-MS/MS, and was determined at a 95% confidence level. We confirmed that the MS/MS spectrum of the peptide derived from secreted decorin was consistent with the decorin spectrum determined in normal tissue. This MRM-based assay demonstrated the high accuracy of target detection by MS/MS analysis (Figure 4c). The absolute quantitations are shown in Figure 4d. We calculated the concentration of decorin in cell-culture medium of siRNA-periostin–treated T47D cells and decorin-overexpressing MDA-MB-231 cells (n = 3). The levels of decorin in medium from siRNA-periostin–treated T47D cells and decorin-overexpressing MDA-MB-231 cells were 0.026 ± 0.007 nM and 0.699 ± 0.143 nM, respectively. The peaks of the endogenous peptide in each control were weak and non-detectable (ND).

Bottom Line: The ability of cancer cells to metastasize is dependent on the interactions between their cell-surface molecules and the microenvironment.However, the tumor microenvironment, especially the cancer-associated stroma, is poorly understood.Our results reveal the molecular details of the periostin-decorin complex in both phyllodes tumor tissues and breast cancer cells; this interaction may represent a novel target for anti-cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University (TMDU) [2] Department of Surgical Oncology, Tokyo Medical and Dental University (TMDU).

ABSTRACT
The ability of cancer cells to metastasize is dependent on the interactions between their cell-surface molecules and the microenvironment. However, the tumor microenvironment, especially the cancer-associated stroma, is poorly understood. To identify proteins present in the stroma, we focused on phyllodes tumors, rare breast tumors that contain breast stromal cells. We compared the expression of proteins between phyllodes tumor and normal tissues using an iTRAQ-based quantitative proteomic approach. Decorin was expressed at reduced levels in phyllodes tumor tissues, whereas periostin was upregulated; this result was validated by immunohistochemical analysis of phyllodes tumors from 35 patients. Additionally, by immunoprecipitation and mass spectrometry, we confirmed that decorin forms a complex with periostin in both phyllodes tumors and BT-20 breast cancer cells. Following siRNA-mediated knockdown of periostin in T-47D cells, secreted decorin in the culture medium could be detected by multiple reaction monitoring (MRM). Furthermore, periostin knockdown in BT-20 cells and overexpression of decorin in MDA-MB-231 cells inhibited cell motility and invasion. Our results reveal the molecular details of the periostin-decorin complex in both phyllodes tumor tissues and breast cancer cells; this interaction may represent a novel target for anti-cancer therapy.

Show MeSH
Related in: MedlinePlus