The Role of Vacuolar Protein Sorting 4 Homolog B in Regulating Epidermal Growth Factor Receptor Signaling in Breast Cancer

Abstract

Breast cancer is the most common cancer among American women. Aberrant signaling by epidermal growth factor receptor (EGFR) is causally involved in the abnormal cell growth, proliferation, invasion and metastasis that occur in breast cancer. Endosomal sorting complexes required for transport (ESCRTs) have been suggested to play an important role in regulating EGFR signaling. Dysfunction of vacuolar protein sorting 4 homolog B (VPS4B), an ESCRT-III associated protein, inhibits EGFR degradation and prolongs intracellular EGFR signaling. Although EGFR signaling is one of the most well characterized signaling pathways, it is not known how changes of the endosomal/lysosomal system can affect EGFR signaling in breast cancer. Here, we analyzed the role of VPS4B-mediated ESCRT dysfunction in altering EGFR signaling in breast cancer cells by a proteomics-based systems biology approach. By using a stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative phosphoproteomics approach coupled with an attenuated VPS4B expression breast cancer cell culture model, we found that EGFR activates epidermal growth factor receptor kinase substrate 8 (EPS8)/SH3 domain-binding protein 1 (SH3BP1) signaling and subsequently stabilizes various cell adhesion and junction molecules. In addition, EGFR activates extracellular signal-regulated kinase (ERK) signaling, and attenuates signal transducer and activator of transcription (STAT) signaling. These results suggest that dysfunction of VPS4B differentially activates and attenuates EGFR signaling in breast cancer cells. An internal standard-assisted synthesis and degradation mass spectrometry (iSDMS) method was developed to determine the consequences of altered EGFR signaling as related to protein synthesis, degradation and relative protein abundance. We found that VPS4B down-regulation results in up-regulation of proteins with roles in fatty acid β-oxidation and down-regulation of proteins involved in glycolysis and fatty acid synthesis. The adoption of fatty acid β-oxidation as an alternative energy source could be an unrevealed survival mechanism for breast cancer cells with VPS4B dysfunction. In summary, our study provides the first systems biology evidence demonstrating the potential pathological relevance of altered EGFR signaling caused by endosomal dysfunction. The study casts a new light on the functional mechanisms underlying breast cancer survival, which will aid the development of new breast cancer treatment strategies.