Proteome analysis of multidrug-resistant, breast cancer–derived microparticles

Figures & data

Fig. 1 Characterization of microparticles (MPs) isolated from MCF-7/Dx cells. a) MPs isolated from MCF-7/Dx cells were analysed via flow cytometry and gated based on sized latex beads (0.3–1.1 µM); b) 34.4% of the gated MP population detected positive for P-gp expression using anti-P-gp mAb (BD Bioscience). All experiments were repeated at least 3 times with similar results. Data are representative of a typical experiment.

Fig. 2 Venn diagram depicting the comparison of proteins identified in microparticles (MPs). Proteins identified in MPs derived from drug-sensitive breast cancer cells (MCF-7) and drug-resistant breast cancer cells (MCF-7/Dx).

Fig. 3 Western blot analysis of selectively packaged proteins in MCF-7-Dx-MPs (microparticles). Twenty micrograms of total cell and MP lysates of drug-sensitive breast cancer cells (MCF-7) and drug-resistant breast cancer cells (MCF-7/Dx) were analysed by Western blot to detect (a) P-glycoprotein, 170 kDa; (b) ezrin, ~80 kDa; (c) moesin, 78–80 kDa; (d) CD44, 82 kDa; and (e) radixin, ~80 kDa. Lanes 1–4 are MCF-7 cells, MCF-7-MPs, MCF-7/Dx cells, and MCF-7/Dx-MPs, respectively. β-actin (42 kDa) was used as the internal control. Data are representative of a typical experiment.

Table I Unique proteins identified in drug-resistant microparticles, which are correlated with cancer and/or multidrug resistance

Protein description	Number of peptide to spectrum matches / (unique peptides)	Percentage of protein coverage	Function in relation to cancer and MDR
Multidrug-resistant 1/P-glycoprotein (P-gp)	37 (33)	30	Energy-dependent efflux pump responsible for decreased drug accumulation in multidrug-resistant cells (4).
CD44	12 (5)	25	Interacts with P-gp to promote cell migration and invasion in cancer. Receptor for hyaluronic acid (HA). Mediates cell–cell and cell–matrix interactions through its affinity for HA, and possibly also through its affinity for other ligands such as osteopontin, collagens and matrix metalloproteinases (MMPs). Adhesion with HA plays an important role in cell migration, tumour growth and progression (37).
5′-nucleotidase	20 (18)	49	Promotes invasion, migration and adhesion of human breast cancer cells. An increased level of protein expression was found in several MDR cell lines, suggesting the involvement of this protein in drug resistance (38, 39).
Glutathione S-transferase P	9 (8)	66	Found to be abundantly expressed in mammalian tissues that are associated with malignancies and play a role in susceptibility to cancer (40).
Ezrin, Radixin and Moesin	34 (12)/11 (3)/7 (4)	43/17/14	Co-immunoprecipitates and co-localizes with P-gp, and helps to interact plasma membrane with cytoskeleton. Probably involved in connections of major cytoskeletal structures to the plasma membrane. In epithelial cells, required for the formation of microvilli and membrane ruffles on the apical pole (22, 41–43).
Aminopeptidase	30 (22)	36	Plays a role in tumour invasion and is found to be elevated in plasma and effusion of cancer patients (44).
Fatty acid binding protein	6 (5)	58	Promotes cancer cell proliferation in hepatoma cells (45).
Talin-1, Vinculin	54 (23)/26 (21)	13/29	Talin 1 interacts with vinculin in order to help in cell adhesion by mediating integrin-mediated adhesion (46).
Phosphoserine aminotransferase	4 (4)	13	Stimulates cell growth and increases chemoresistance of colon cancer cells (47).
Stathmin	3 (2)	27	Controls cell proliferation and participates in carcinogenesis of various carcinomas. Involved in the regulation of the microtubule (MT) filament system by destabilizing microtubules. Prevents assembly and promotes disassembly of microtubules (48).
Filamin	134 (61)	33	Promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. Anchors various transmembrane proteins to the actin cytoskeleton and serves as a scaffold for a wide range of cytoplasmic signalling proteins. Interaction with FLNA may allow neuroblast migration from the ventricular zone into the cortical plate (49).
Alpha-actinin-4	41 (35)	50	Regulates the actin cytoskeleton and increases cellular motility; its inactivation by transfer to the nucleus abolishes the metastatic potential of human cancers (50).
Fermitin family homolog 2	6 (4)	10	Enhances integrin-mediated cell adhesion onto the extracellular matrix and cell spreading (51).

Proteins identified from MPs derived from drug-resistant breast cancer cells, which are reported in the literature to directly or indirectly contribute to cancer and/or MDR. The list is generated with the total unique peptide count and the protein coverage to determine the percentage of the residues in each protein sequence.

Fig. 4 Protein network generated from String 9.05. Network of unique proteins associated with ABCB1/P-gp, isolated from microparticles derived from drug-resistant breast cancer cell (MCF-7/Dx), visualized on the String website. Proteins such as CD44, tropomyosin 3, glutathione S-transferase P1 and ecto-5′-nucleotidase are associated with drug-resistant proteins, whereas fibronectin 1, intercellular adhesion molecule 1, ezrin, moesin, integrin beta 3, vinculin, basigin, rho family and vimentin are associated with CD44.

Fig. 5 Microparticles (MPs) play a deleterious role in cancer by transferring drug-resistant proteins. MP surface molecules (adhesion molecules) and FERM domain proteins are proposed to be associated with tissue selectivity in the transfer of P-gp in malignant breast cells.

Table II Pathway analysis of proteins unique to resistant breast cancer–derived microparticles

GO_id	Pathway	Number of proteins	p	p-value fdr	p-value Bonferroni
hsa03010	Ribosome	25	2.94 E−24	6.23 E−22	6.23 E−22
hsa04810	Regulation of actin cytoskeleton	10	6.62 E−4	7.02 E−2	1.4 E−1
hsa04510	Focal adhesion	8	8.54 E−3	4.53 E−1	1 E−0
hsa04670	Leukocyte transendothelial migration	7	1.21 E−3	8.55 E−2	2.56 E−1
hsa04520	Adherens junction	4	2.27 E−2	9.65 E−1	1 E−0

Top 5 pathways were generated with the help of KEGG pathway analysis. Proteins related to regulation of actin cytoskeleton, focal adhesion, ECM–receptor interaction and others were predicted to play a significant role in transferring p-gp and cause of drug resistance in cells.

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