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

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.


Supplementary
. Decorin is upregulated in normal tissues relative to tumor tissues, whereas periostin is upregulated in tumor tissues. Normal and tumor tissues from three phyllodes tumor patients were depleted of albumin and subjected to trypsin digestion. Tryptic peptides were labeled using the iTRAQ reagents. Normal tissue lysates were tagged with isobaric tags with m/z of 114, and tumor tissue lysates were tagged with isobaric tags with m/z of 117. The tagged lysates were mixed and separated by chromatography on a C 18 capillary column using elution buffer containing KCl (10,25,50,100,175,and 350 mM). The six fractions were analyzed by mass spectrometry in at least three trials.   Supplementary Figure S4 Supplementary Figure S4. MRM chromatograms for DLPPDTTLLDLQNNK fragments and their standard (AQUA: red line) analogues. MRM chromatograms for VSPGAFTPLVK was shown in Figure 4a. The peptides in culture medium from siRNAperiostin-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. Figure S5. MRM chromatograms for VSPGAFTPLVK fragments and their standard (AQUA) analogues. Two MRM transitions for the endogenous (blue line) and standard (red line) peptides were monitored( Figure 4b). For VSPGAFTPLVK, the doubly charged precursor mass was chosen as the Q1 mass, and the y9 fragment ions were chosen as Q3 mass. Insets contain magnified views. MRM-triggered MS/MS product ion spectra obtained by nanoflow LC/MS/MS, comparing normal tissue digested with trypsin (Firgure 4c; upper) with decorin secreted from decorin-overexpressing MDA-MB-231 cells (Figure 4c; lower). The spectrum of the peptide clearly shows y-ion fragments.    Immunoblotting analysis Tissue and cell lysates were subjected to SDS-PAGE and transferred electrophoretically onto Immobilon-P transfer membrane (EMD Millipore, Billerica, MA, USA). The membrane was incubated in SuperBlock Blocking Buffer (Thermo Scientific, Waltham, MA, USA) at room temperature (RT) for 1 h, stained with primary antibodies for 2 h at RT, and incubated for 1 h at RT with secondary antibodies coupled to horseradish peroxidase (ECL anti-mouse or anti-rabbit IgG; Amersham/GE Healthcare, Buckinghamshire, UK). The blots were developed using the SuperSignal enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA) and exposed to Kodak X-OMAT film. All antibodies are listed in Supplementary Table S4. As needed, protein bands were quantitated using the ImageJ software (version 1.46r, National Institutes of Health, Bethesda, MD, USA).

Immunoprecipitation
Tissue and cell lysates were immunoprecipitated for 30 min at 4°C with 5 μg antibody, followed by incubation for 30 min at 4°C with Dynabeads-Protein G (Invitrogen, Carlsbad, CA, USA). Immune complexes were precipitated and washed. Immunoprecipitated proteins were loaded to onto e-PAGEL precast gels (ATTO, Tokyo, Japan) for mass spectrometry or subjected to standard SDS-PAGE for immunoblotting.
Knockdown of gene expression siRNAs specifically targeting periostin and decorin were purchased from Dharmacon (Lafayette, CO, USA. For transfections, Lipofectamine RNAiMAX (Invitrogen) and 12 nM siRNA were mixed in 2 ml Opti-MEM (Life Technologies) and incubated for 20 minutes at RT; the mixture was then added onto 70% confluent cultures. After 72 h, RNA and protein were isolated.
Total RNA extraction and RT-PCR Total RNA was extracted from the lysates BT-20 using the QuickPrep Micro mRNA Purification Kit (GE Healthcare, Little Chalfont, UK). Decorin full-length cDNA was generated from RNA by RT-PCR using the SuperScript III One-Step RT-PCR System with Platinum Taq (Life Technologies) and subcloned into the pME18S-HA-C vector in frame with the appropriate N-terminal tags. Clonal cell lines were obtained by transfection of 2 μg of plasmid DNA using the Lipofectamine Plus transfection reagent (Invitrogen). Reverse transcription was performed in a reaction volume of 60 ml containing 90 ng of total RNA and 2 ml of SuperScript III One-Step RT-PCR System with Platinum Taq. The conditions for polymerase chain reaction (PCR) were as follows: 94°C for 2 min; 40 cycles of 94°C for 15 s, 60°C for 30 s, and 68°C for 90 s; and a final cycle of 68°C for 5 min. The PCR products were electrophoresed on 1% agarose gels. The PCR primers for DCN were 5'-CCGGAATTCATGAAGGCCACTATCATCC-3' and 5'-GCCGCTCGAGTTACTTATAGTTTCCGAG-3'.

Immunofluorescence
Cells were seeded onto chamber slides (Matsunami, Osaka, Japan) and fixed with 3.5% formaldehyde in PBS for 10 minutes on ice. Fixed cells were permeabilized with 50%, 75%, and 95% cold ethanol on ice for 5 minutes each. Fixed cells were blocked for 30 min, incubated with primary antibody for 1 h at RT, incubated with Alexa Fluor 488-or Alexa Fluor 594-conjugated secondary antibody (Molecular Probes, Life Technologies) for 30 min at 37°C, and then preserved in Vectashield (Vector Laboratories, Burlingame, CA, USA). DNA was stained with bisbenzimide (Hoechst 33258, Life Technologies). The samples were examined with an Olympus Power BX51 fluorescence microscope (Olympus, Tokyo, Japan). Confocal microscopy was performed using a Leica TS SP8, and the confocal images were analyzed using the Leica SP8 LAS AF Lite software (Leica, Solms, Germany).
Cell proliferation assay Cells were seeded in 96-well flat-bottomed microtiter plates and cultured for 24 h, followed by incubation for 30 minutes with Cell Proliferation Reagent WST-1 (Roche). Cleavage of WST-1 to formazan by metabolically active cells was quantitated by scanning the plates at 450 nm and 650 nm (reference wavelength) in a microtiter plate reader. Viability of treated cells was normalized to that of untreated control cells.