Dataset Information

Identification of phosphorylation sites regulated by lysophosphatidic acid (LPA) in OCMI91s ovarian cancer cells

ABSTRACT: 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 as described above and lysed in 100 mM Tris pH 7.6, 4% SDS, PhosSTOP (Roche, #4906845001), Protease Inhibitor Cocktail (Sigma, P8340). Analysis of OCMI91s lysates were performed as described [PMID: 19029910]. The protocol was modified to use 12 ug of peptide per channel for TMT labeling, resulting in 500 ug multiplexed samples. LC-MS2 analysis was performed on 50% of the corresponding sample material rehydrated in 0.1% formic acid as published [PMID: 19029910]. Detailed information on instrumentation parametrization was extracted and summarized using MARMoSET [PMID: 310976732] 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 (versions 1.6.8.0, [PMID: 19029910]) against the human canonical and isoforms Uniprot database (downloaded 20190819, 173199 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 >1.2x or <0.83x and p <0.1.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Rolf Mueller

PROVIDER: MSV000091146 | MassIVE | Wed Jan 25 09:13:00 GMT 2023

SECONDARY ACCESSION(S): PXD039689

REPOSITORIES: MassIVE

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The lysophosphatidic acid-regulated signal transduction network in ovarian cancer cells and its role in actomyosin dynamics, cell migration and entosis.

Ojasalu Kaire K Lieber Sonja S Sokol Anna M AM Nist Andrea A Stiewe Thorsten T Bullwinkel Imke I Finkernagel Florian F Reinartz Silke S Müller-Brüsselbach Sabine S Grosse Robert R Graumann Johannes J Müller Rolf R

Theranostics 20230321 6

Lysophosphatidic acid (LPA) species accumulate in the ascites of ovarian high-grade serous cancer (HGSC) and are associated with short relapse-free survival. LPA is known to support metastatic spread of cancer cells by activating a multitude of signaling pathways via 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. <b>Methods:</b> LPA-induced signaling pa ...[more]

PMID: 37064875

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Project description: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 >1.2x or <0.83x and p <0.1.

Project description:Background: Transcoelomic spread is the major route of metastasis of ovarian high-grade serous carcinoma (HGSC) with the omentum as the major metastatic site. Its unique tumor microenvironment with its large populations of adipocytes, mesothelial cells and immune cells establishes an intercellular signaling network that is instrumental for metastatic growth yet poorly understood. Methods: Based on transcriptomic analysis of tumor cells, tumor-associated immune and stroma cells we defined intercellular signaling pathways for 284 cytokines and growth factors and their cognate receptors after bioinformatic adjustment for contaminating cell types. The significance of individual components of this network was validated by analyzing clinical correlations and potentially pro-metastatic functions, including tumor cell migration, pro-inflammatory signal transduction and TAM expansion. Results: The data show an unexpected prominent role of host cells, and in particular of omental adipocytes, mesothelial cells and fibroblasts (CAF), in sustaining this signaling network. These cells, rather than tumor cells, are the major source of most cytokines and growth factors in the omental microenvironment (n=176 versus n=13). Many of these factors target tumor cells, are linked to metastasis and are associated with a short survival. Likewise, tumor stroma cells play a major role in both extracellular-matrix-triggered and lipid-mediated signaling. We have verified the functional significance of our observations for three exemplary instances. We show that the omental microenvironment (i) stimulates tumor cell migration via WNT4 which is highly expressed by CAF; (ii) induces pro-tumorigenic TAM proliferation in conjunction with high CSF1 expression by omental stroma cells and (iii) triggers pro-inflammatory signaling, at least in part via a HSP70 – NFkB pathway. Conclusions: The intercellular signaling network of omental metastases is majorly dependent on factors secreted by immune and stroma cells to provide an environment that supports ovarian HGSC progression. Clinically relevant pathways within this network represent novel options for therapeutic intervention.

Project description:The fallopian tube epithelium is one of the potential sources of high-grade serous ovarian cancer (HGSC). The use of estrogen only hormone replacement therapy increases ovarian cancer risk. Despite estrogenâ??s influence in OVCA, selective estrogen receptor modulators (SERMs) typically demonstrate only a 20% response rate. This low response could be due to a variety of factors including the loss of estrogen receptor signaling or the role of estrogen receptor signaling in different potential cell types of origin. The response of fallopian tube epithelium to SERMs is not known, and would be useful when determining therapeutic options for tumors that arise from this cell type, such as high-grade serous cancer. Using normal murine derived oviductal epithelial cells (mouse equivalent to the fallopian tube) estrogen receptor expression was confirmed and interaction with its ligand, estradiol, triggered mRNA and protein induction of progesterone receptor (PR). The SERMs 4-hydroxytamoxifen, raloxifene and desmethylarzoxifene, functioned as estrogen receptor antagonists in the oviductal cells. Cellular proliferation and migration assays suggested that estradiol does not significantly impact cellular migration and increased proliferation in CD1, but not in FVB derived cell lines. Further, using RNAseq, the oviduct specific transcriptional genes targets of estrogen and 4-hydroxytamoxifen signaling were determined and validated. The RNA-seq revealed enrichment in proliferation, anti-apoptosis, calcium signaling and steroid signaling processes. Finally, the ER and PR receptor status of a panel of HGSC cell lines was investigated highlighting the need for better models of estrogen responsive HGSC cell lines. Murine oviductal epithelial cells from the FVB background were hormone starved for 48 hours (with a media change after 24 hours), then treated in triplicate with solvent control (DMSO) (0.1%), 1 nM 17-betaestradiol or 100 nM 4-hydroxytamoxifen for 24 hours. Following treatment, RNA was was isolated, libraries were prepped and sequenced using the Illumina HiSeq 2500 platform.