Correlated S-palmitoylation profiling of Snail-induced epithelial to mesenchymal transition

Abstract

Epithelial cells form spatially-organized adhesion complexes that establish polarity gradients, regulate cell proliferation, and direct wound healing. As cells accumulate oncogenic mutations, these key tumor suppression mechanisms are disrupted, eliminating many adhesion complexes and bypassing contact inhibition. The transcription factor Snail is often expressed in malignant cancers, where it promotes transcriptional reprogramming to drive epithelial-mesenchymal transition (EMT), which promotes a more invasive state. S-palmitoylation describes the fatty-acyl post-translational modification of cysteine residues in proteins, and is required for membrane anchoring, trafficking, localization and function of hundreds of proteins involved in cell growth, polarity, and signaling. Since Snail-expression prevents apico-basolateral cell polarity, we asked if Snail-dependent transformation induces proteome-wide changes in S-palmitoylation. MCF10A breast cancer cells were retrovirally transduced with Snail, and correlated proteome-wide changes in protein abundance and S-palmitoylation were profiled using by stable isotope labeling in cell culture with amino acids (SILAC) mass spectrometry. This analysis identified increased levels of proteins involved in migration, glycolysis, and cell junction remodeling, and decreased levels of proteins involved in cell adhesion. Overall, protein S-palmitoylation is highly correlated with protein abundance, yet for a subset of proteins, this correlation is uncoupled. These findings suggest that Snail-overexpression affects the S-palmitoylation cycle of some proteins, and may affect cell polarity and tumor suppression.

APT2 Inhibition Restores Scribble Localization and S -Palmitoylation in Snail-Transformed Cells

“…Since Scrib requires S -palmitoylation for membrane recruitment (Chen et al, 2016), we explored if defects in Scrib S -palmitoylation might provide a potential mechanism for mislocalization during malignancy. Polarized MDCK and MCF10A epithelial cells were transduced with the EMT-TF Snail, which transcriptionally represses the expression of cellular adhesion proteins like E-cadherin, while activating expression of antioxidant, glycolytic, and cytoskeletal remodeling enzymes (Hernandez et al, 2016). In control cell lines transduced with an empty viral vector, Scrib and E-cadherin co-localize at the plasma membrane, confirming a polarized phenotype (Qin et al, 2005).…”

“…To explore this question, total RNA was isolated from MCF10A control and MCF10A-Snail cells for quantitative, real-time PCR (RT-qPCR) of all 23 zDHHC PATs. RT-qPCR values were normalized to β-actin, which was previously validated by mass spectrometry to remain constant after Snail over-expression (Hernandez et al, 2016). Surprisingly, nearly all zDHHC mRNAs were significantly reduced in expression after Snail over-expression, including an 8-fold reduction in zDHHC23 expression (Figure 2A and Figure S1).…”

“…This is also corroborated by Oncomine expression analysis, which classifies APT2 as a significantly up-regulated gene in breast cancer (Rhodes et al, 2004). Indeed, Snail over-expression in MCF10A led to a 2-fold or greater reduction in alkynyl-fatty acid bioorthogonal enrichment for ~13% of validated S -palmitoylated proteins, which was often uncoupled from protein abundance (Hernandez et al, 2016). This targeted suppression of zDHHC enzymes demonstrates Snail functionally targets the S -palmitoylation machinery, which cooperates in disrupting cell polarity and the displacement of Scrib from the plasma membrane.…”

“…We previously identified Scrib as palmitoylated in MCF10A human epithelial cells (Hernandez et al, 2016), as well as the most dynamic and enzymatically regulated palmitoylated protein in highly aggressive T-cell hybridoma cells (Martin et al, 2012). Indeed, the SwissPalm database lists Scrib as one of the more detectable palmitoylated proteins in mammalian cells, returning confident annotations in 12 of 25 reported mammalian S -palmitoylation proteomics datasets (Blanc et al, 2015).…”

Chemoselective ratiometric imaging of protein S-sulfenylation

“…The transcription factor Snail (SNAI1) is elevated in metastatic cancers, and plays a key role in reprogramming cells to undergo epithelial-to-mesenchymal transition (EMT) through the loss of the cellular adhesion protein E-cadherin 27 . Quantitative mass spectrometry profiling of Snail-expressing cells recently demonstrated a >3-fold elevation of many antioxidant enzymes, including peroxiredoxins, superoxide dismutase, and thioredoxin 28 . Based on these data, we examined if Snail overexpression affects redox homeostasis to influence protein S -sulfenylation levels in live cells.…”