Rat mammary extracellular matrix composition and response to ibuprofen treatment during postpartum involution by differential GeLC-MS/MS analysis

Abstract

Breast cancer patients diagnosed within five years following pregnancy have increased metastasis and decreased survival. A hallmark of postpartum biology that may contribute to this poor prognosis is mammary gland involution, involving massive epithelial cell death and dramatic stromal remodeling. Previous studies show pro-tumorigenic properties of extracellular matrix (ECM) isolated from rodent mammary glands undergoing postpartum involution. More recent work demonstrates systemic ibuprofen treatment during involution decreases its tumor-promotional nature. Utilizing a proteomics approach, we identified relative differences in the composition of mammary ECM isolated from nulliparous rats and those undergoing postpartum involution, with and without ibuprofen treatment. GeLC-MS/MS experiments resulted in 20327 peptide identifications that mapped to 884 proteins with a <0.02% false discovery rate. Label-free quantification yielded several ECM differences between nulliparous and involuting glands related to collagen-fiber organization, cell motility and attachment, and cytokine regulation. Increases in known pro-tumorigenic ECM proteins osteopontin, tenascin-C, and laminin-α1 and pro-inflammatory proteins STAT3 and CD68 further identify candidate mediators of breast cancer progression specific to the involution window. With postpartum ibuprofen treatment, decreases in tenascin-C and three laminin chains were revealed. Our data suggest novel ECM mediators of breast cancer progression and demonstrate a protective influence of ibuprofen on mammary ECM composition.

Figure 1. Transformed mammary epithelial cells cultured with mammary ECM from rats treated systemically with ibuprofen for 6 days post-weaning phenocopy cells cultured in quiescent mammary ECM from nulliparous rats

Brightfield images of Ras-transformed MCF12A cells in 3D culture at 48 hours. (Coating assay, ECM at 200 μg/ml, images 50X)

Figure 2. Relative abundance of select proteins based on normalized peak area for all peptides assigned per band

A) Colloidal coomassie blue stained SDS-PAGE image of matrix isolated from nulliparous, treated animals with approximate molecular weights based on molecular weight markers. Approximate location of the 17 gel bands is shown. Peptide identifications throughout the 17 bands for A) Albumin (ALB), B) Whey acidic protein (WAP), C) κ-casein (CSN3), D) laminin α1 (LAMA1), E) laminin β1 (LAMB1), F) laminin β2 (LAMB2), G) collagen VII α1 (COL7A1), H) collagen XVII α1 (COL17A1), and I) dermatopontin (DPT). Calculated ion intensities normalized for each replicate to max intensity from the four samples. Nul=nulliparous, Inv=control involution, InvIb=ibuprofen involution

Figure 3. Estimation of protein abundance based on spectral counts

Spectral counts were used to estimate the percentage of proteins from a given cellular component; results are presented for the top 25, next 75, 150, 250 and 488 proteins based on peptide counts. N=nulliparous, I=control involution, IIb=ibuprofen involution

Figure 4. Protein-protein interaction network for proteins identified in our tandem mass spectrometry runs

ECM components and select ECM receptors were submitted for analysis of protein interactions (using STRING-DB.org). Components at the cell membrane are in the upper left corner, basement membrane moving down and right toward the fibrillar collagens at the bottom. Immune cell related proteins discussed in the text are diagramed in the upper right on a macrophage.