An Internal Standard-Assisted Synthesis and Degradation Proteomic Approach Reveals the Potential Linkage between VPS4B Depletion and Activation of Fatty Acid β-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.

Figure 1

Using internal standard-assisted synthesis and degradation mass spectrometry (iSDMS) to study the roles of EGF on global protein synthesis and degradation. SKBR3_shVPS4B and the parental SKBR3 cells were first labeled with ¹³C₆-arginine (Arg6) and D₄-lysine (Lys4) medium (labeled in red). After overnight serum starvation, Arg6/Lys4-labeled cells were stimulated with 100 ng/mL EGF in medium supplemented with regular arginine (Arg0) and lysine (Lys0) for 0, 2, 6, 12, and 24 hr (labeled in blue). Protein isolated from SKBR3_shVPS4B cells labeled with ¹³C₆ ¹⁵N₄-arginine (Arg10) and ¹³C₆ ¹⁵N₂-lysine (Lys8)—internal standard (labeled in green)—was spiked into each sample at a ratio of 1 : 3 (wt/wt). The mixtures were digested by the Filter Aided Sample Preparation (FASP) procedure, followed by strong anion exchange (SAX) peptide fractionation. Peptides were analyzed by online LC-MS/MS using an LTQ-Orbitrap mass spectrometer. The relative abundance of the newly synthesized (A _l) or preexisting peptides (A _m) was defined as the ratio of mass spectrometric peak intensities of the unlabeled peptides (I _l) or Arg6/Lys4-labeled peptides (I _m) to the intensities of the Arg10/Lys8-labeled peptides (I _h), respectively.

Figure 2

VPS4B downregulation decreases the expression of fatty acid synthase (FASN) in SKBR3_shVPS4B cells. Representative MS1 spectra of fatty acid synthase peptide SLLVNPEGPTLMR in SKBR3 (a) and SKBR3_shVPS4B cells (b). The relative abundance of unlabeled peptides (A _l, labeled in blue) or labeled peptides (A _m, label in red), expressed as mean ± standard  deviation, was calculated by our in-house software, IsoQuant [38]. VPS4B downregulation increased the degradation rate of the SLLVNPEGPTLMR FASN peptide (c) and decreased its synthesis rate (d) in SKBR3 cells, which was related to the decrease of its relative abundance after EGF treatment (e).

Figure 3

The role of VPS4B downregulation on the alteration of global protein synthesis and degradation after EGF treatment. Ratios of synthesis rates of SKBR3_shVPS4B versus SKBR3 cells (y axis) are plotted against ratios of degradation rate constants of SKBR3_shVPS4B versus SKBR3 cells (x axis). Diamonds represent log⁡₂ ratios of protein expression at 24 hr between the two cell lines, and the relative values of which are indicated on the color scale.

Figure 4

VPS4B downregulation increases the expression of mitochondrial fatty acid β-oxidation related proteins and down regulates the expression of glycolysis related proteins in SKBR3 cells. The energy metabolism pathway was adapted from the Kyoto Encyclopedia of Genes and Genomes (KEGG) [40]. Colors represent the ratios of relative protein abundance at 24 hr (shaded ellipses), protein synthesis rate (shaded upper rectangles), and protein degradation rate constant (shaded lower rectangles) in SKBR3_shVPS4B versus SKBR3 cells. Red color represents protein with increased expression and green color represents protein with decreased expression.