An Internal Standard-Assisted Synthesis and Degradation Proteomic Approach Reveals the Potential Linkage between VPS4B Depletion and Activation of Fatty Acidβ-Oxidation in Breast Cancer Cells

Abstract

The endosomal/lysosomal system, in particular the endosomal sorting complexes required for transport (ESCRTs), plays an essential role in regulating the trafficking and destination of endocytosed receptors and their associated signaling molecules. Recently, we have shown that dysfunction and down-regulation of vacuolar protein sorting 4B (VPS4B), an ESCRT-III associated protein, under hypoxic conditions can lead to the abnormal accumulation of epidermal growth factor receptor (EGFR) and aberrant EGFR signaling in breast cancer. However, the pathophysiological consequences of VPS4B dysfunction remain largely elusive. In this study, we used an internal standard-assisted synthesis and degradation mass spectrometry (iSDMS) method, which permits the direct measurement of protein synthesis, degradation and protein dynamic expression, to address the effects of VPS4B dysfunction in altering EGF-mediated protein expression. Our initial results indicate that VPS4B down-regulation decreases the expression of many proteins involved in glycolytic pathways, while increased the expression of proteins with roles in mitochondrial fatty acid β-oxidation were up-regulated in VPS4B-depleted cells. This observation is also consistent with our previous finding that hypoxia can induce VPS4B down-regulated, suggesting that the adoption of fatty acid β-oxidation could potentially serve as an alternative energy source and survival mechanism for breast cancer cells in response to hypoxia-mediated VPS4B dysfunction.

A splicing mutation inVPS4Bcauses dentin dysplasia I

“…In addition, VPS4B has been shown to interact with the endosomal membrane to regulate the internalisation and lysosomal degradation of membrane proteins,30 and it also participates in intracellular protein trafficking, abscission of cytokinesis and virus budding 31. Depletion of VPS4B protein causes defects of nuclear morphology and chromosome segregation 19.…”

The role of the endosomal sorting complexes required for transport (ESCRT) in tumorigenesis

“…Depletion by sh RNA or expression of a dominant negative Vps4 mutant blocked EGFR degradation and enhanced EGFR signaling, which in turn promoted anchorage-independent growth and resistance to Gefitinib , a tyrosine kinase inhibitor mainly affecting EGFR kinases (Lin et al, 2012 ). Interestingly, the glycolytic pathway is down-regulated in VPS4B-depleted human mammary gland SKBR3 cells, suggesting a potential crosstalk between the abnormal metabolism of cancer cells and MVB dysfunction (Liao et al, 2013 ). Overall, it seems that neither in vivo model organisms nor in vitro cell culture experiments offer a simple or unifying answer to the role of the ESCRT machinery in tumorigenesis.…”

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

“…The energy that is required for the concomitant dissociation of the ESCRT machinery and scission of the ILV from the endosomal membrane is provided by the multimeric protein complex known as the VPS4 complex. Studies from our laboratory using the HER2 over-expressing breast cancer cell line SKBR3 have demonstrated that hypoxia or reactive oxygen species (ROS)-induced VPS4 dysregulation leads to the accumulation of the ESCRT machinery in exosomes, as well as an increase in proteins that are associated with exosome biogenesis and trans-Golgi network receptor recycling [ 38 , 54 , 55 ] ( Figure 3 ). Furthermore, under these conditions, an accumulation of exosome protein cargo was observed, which resulted in the increased relative abundance of EGFR and HER2 receptors.…”