Exosomal Proteome Profiling: A Potential Multi-Marker Cellular Phenotyping Tool to Characterize Hypoxia-Induced Radiation Resistance in Breast Cancer

Abstract

Radiation and drug resistance are significant challenges in the treatment of locally advanced, recurrent and metastatic breast cancer that contribute to mortality. Clinically, radiotherapy requires oxygen to generate cytotoxic free radicals that cause DNA damage and allow that damage to become fixed in the genome rather than repaired. However, approximately 40% of all breast cancers have hypoxic tumor microenvironments that render cancer cells significantly more resistant to irradiation. Hypoxic stimuli trigger changes in the cell death/survival pathway that lead to increased cellular radiation resistance. As a result, the development of noninvasive strategies to assess tumor hypoxia in breast cancer has recently received considerable attention. Exosomes are secreted nanovesicles that have roles in paracrine signaling during breast tumor progression, including tumor-stromal interactions, activation of proliferative pathways and immunosuppression. The recent development of protocols to isolate and purify exosomes, as well as advances in mass spectrometry-based proteomics have facilitated the comprehensive analysis of exosome content and function. Using these tools, studies have demonstrated that the proteome profiles of tumor-derived exosomes are indicative of the oxygenation status of patient tumors. They have also demonstrated that exosome signaling pathways are potentially targetable drivers of hypoxia-dependent intercellular signaling during tumorigenesis. This article provides an overview of how proteomic tools can be effectively used to characterize exosomes and elucidate fundamental signaling pathways and survival mechanisms underlying hypoxia-mediated radiation resistance in breast cancer.

Keywords: breast cancer; exosomes; hypoxia; proteomics; radiation; tumor microenvironment.

Figure 1

The hypoxia-induced release of exosomes from cancer cells is hypothesized to result in the malignant transformation and subsequent proliferation and migration of normal recipient cells.

Figure 2

Spatial relationship between a blood vessel, hypoxic conditions and a malignant solid tumor in the context of O₂ and HIF-1 concentrations. Normoxic conditions, which are defined by oxygen concentrations above 5%, exist in regions that are less than 70 µM from tumor blood vessels. In these regions, HIF-1 is rapidly degraded, or it has a low level of expression. Hypoxic conditions exist at distances greater than 100 µM from tumor blood vessels. In these regions, HIF-1 expression is increased and O₂ concentrations are decreased, which creates a hypoxic tumor microenvironment.

Figure 3

Increased exosome release resulting from hypoxia or reactive oxygen species (ROS)-induced VPS4 dysregulation.

Figure 4

Workflow for stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic profiling of exosomal proteins. Cell lines are cultured in SILAC media that has been supplemented with arginine and lysine containing ¹³C and ¹⁵N (Lys8, Arg10; “heavy”) or the naturally occurring ¹²C and ¹⁴N isotopes (Lys0, Arg0; “light”). After exposure to hypoxic or normoxic conditions, the exosomes are isolated from each cell line and are mixed at a 1:1 ratio followed by enzymatic protein digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

Figure 5

Representative SILAC MS1 spectra of Alix, CD63 and HER2 peptides with decreased relative abundance in exosomes that are derived from SKBR3 cells with ablated VPS4B expression. The relative abundance ratios were determined by IsoQuant.

Figure 6

Immunoblot analysis shows accumulation of GSK3α/β and phosphorylated ERK2 signaling pathway proteins such as RSK1 in exosomes derived from SKBR3 cells with decreased VPS4B expression. Hexokinase-1 was included as an immunoblotting control for the specificity of the effect of decreased VPS4B expression. The level of hexokinase-1 expression was unchanged between exosomes from SKBR3 cells expressing WT VPS4B and cells with shRNA-mediated knockdown of VPS4B expression (VPS4B KD).

Figure 7

Proposed breast cancer signaling pathway based on the KEGG network pathway analysis of hypoxia-activated tumor cell migration. In EGF-stimulated SKBR3 cells with decreased VPS4B expression, the assembly of F-actin and the formation of filopodia were promoted, suggesting that the dysfunction of the MVB pathway stimulates the migration of tumor cells under hypoxic conditions.