DOI: 10.25345/c5pc2tk19
title: MassIVE MSV000091158 - Identification of phosphorylation sites regulated by lysophosphatidic acid (LPA) in OVCAR8 ovarian cancer cells
title: Elevated levels of lysophosphatidic acid (LPA) species accumulate in the ascites of ovarian high grade serous cancer (HGSC) and are associated with a short relapse-free survival. LPA is known to support the metastatic spread of cancer cells by activating a multitude of signaling pathways through G-protein-coupled receptors of the LPAR family. Systematic unbiased analyses of the LPA-regulated signal transduction network in ovarian cancer cells have, however, not been reported to date. Methods: LPA-induced signaling pathways were identified by phosphoproteomics of both patient-derived cells and the HGSC cell line OVCAR8, RNA sequencing, pharmacological inhibition, measurements of intracellular Ca2+ and cAMP levels as well as cell imaging. The function of LPARs and downstream signaling components in migration and the formation of entotic cell-in-cell structures was analyzed by applying selective pharmacological inhibitors and RNA interference. Results: Transcriptional signaling by LPA is mainly mediated by LPAR1 to target genes promoting cell motility and migration via multiple cooperating pathways, including PKC, PKD1 and ERK1/2, as demonstrated for IL6 and THBS1 genes. Likewise, cytoplasmic signaling targeting actomyosin is mediated by interconnected phosphorylation-driven pathways triggered prominently by LPAR1. A central component of these signaling pathways is the protein phosphatase 1 regulatory subunit MYPT1, which is a negative regulator of myosin light chain 2 (MYL2). MYPT1 is negatively regulated by PKC- and ERK-mediated phosphorylation in response to LPA, and is indispensable for LPA-induced actomyosin-dependent cell migration and entosis (cell-in-cell invasion). We further show a novel LPAR2-DOCK7 pathway to be essential for the induction of entosis. Conclusion: The LPAR1-ERK/PKC-MYPT1 axis is a critical pathway of LPA-triggered cytoskeletal changes, cell migration and entotic cell-in-cell invasion, pointing to MYPT1 as a promising candidate for therapeutic intervention. For phosphoproteomic analyses cells were treated with LPA, antagonists or solvent at least in triplicate and lysed in 100 mM Tris pH 7.6, 4% SDS, PhosSTOP (Roche, #4906845001), Protease Inhibitor Cocktail (Sigma, P8340). Analysis of OVCAR8 lysates proceeded as follows: samples were subjected to acetone precipitation and following tryptic digest of 600 ug total protein per sample as described [PMID: 33391540], as well as desalting using Waters Oasis HLB cartridges (Waters, #186006339) and peptide yield determination using the Quantitative Fluorometric Peptide Assay (Pierce, #23290), each sample was labeled using TMTsixplex (Thermo Scientific, #90064) following the manufacturer's protocol. Subsequent to mass spectrometric validation of labeling efficiency and mixing 80 ug peptides per channel using samples from solvent control experiments as bridging samples where more than a single sixplex was required to cover replicate experiments, multiplexed samples were desalted once more as described above. Of the resulting pooled samples, 30 ug each where fractionated into eight fractions using High pH Reversed-Phase Peptide Fractionation Kit (Pierce, #84868) using the manufacturer's protocol and evaporation to dryness for subsequent analysis of the background proteome. In the remaining multiplexed peptide samples, phosphorylated peptides where enriched using the High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific, #A32992). While 5% (10 ul) of the resulting eluate where evaporated to dryness for direct subsequent liquid chromatography/tandem mass spectrometry (LC-MS2) analysis, the remainder was once more separated into eight fractions using the High pH Reversed-Phase Peptide Fractionation Kit and fractions subsequently evaporated to dryness. LC-MS2 analysis was performed on 50% of the corresponding sample material rehydrated in 0.1% formic acid as published [PMID: 33391540]. Detailed information on instrumentation parametrization was extracted and summarized using MARMoSET [PMID: 31097673] and is included in the repository-deposited data set. Peptide/spectrum matching as well as TMT quantitation was performed using the MaxQuant suit of algorithms (version 2.0.3.0 [PMID: 19029910]) against the human canonical and isoforms Uniprot database (downloaded 20211026, 202160 entries). Mass spectrometric raw data along with documentation of instrumentation parameters governing its acquisition as well as MaxQuant parameters employed are deposited. Data were filtered for rows containing no zero values, and analyzed by paired Student's t-test (blocking on replicate set / phosphoproteome analysis chip). Thresholds were set according to the dynamic range of the assay to FC &gt;1.2x or &lt;0.83x and p &lt;0.1.