Dataset Information

PI3K-AKT pathway and cell-cell contact regulates maspin nuclear localization

ABSTRACT: Background: Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies indicate that nuclear localization is essential for maspin tumor suppression activity. We have previously shown that the EGFR activation leads to maspin nuclear localization in MCF-10A cells. The present study investigated which EGFR downstream signaling molecules are involved in maspin nuclear localization and explored a possible role of cell-cell contact in this process. Methods: MCF-10A cells were treated with pharmacological inhibitors against EGFR downstream pathways followed by EGF treatment. Maspin subcellular localization was determined by immunofluorescence. To investigate the role of cell-cell contact these cells were either treated with chelating agents or plated on different cell densities. Maspin and E-cadherin subcellular localization was determined by immunofluorescence. In addition, proteomic and interactome analyses were conducted in order to identify molecular partners which interact with maspin in EGF-treated cells only. Results: EGFR activation regulates cell behavior via de extracellular signal–related kinase (ERK)/MAP kinase, phosphatidyl-inositol-3 (PI 3) kinase- AKT and Jak-STAT pathways. Using pharmacological inhibitors against key components of these signaling pathways we found that PI3K-AKT and Jak-STAT, but not MAP kinase pathway, regulate EGF-induced maspin nuclear accumulation in MCF-10A cells. Interestingly, we observed that maspin is predominantly nuclear in sparse cell culture, but it is redistributed to the cytoplasm in confluent cells even in the presence of EGF. Proteomic and interactome results suggest a role of maspin on post-transcriptional and translation regulation, protein folding and cell-cell adhesion.Conclusions: Maspin nuclear accumulation is determined by an interplay between EGFR (via PI3K-AKT and Jak-STAT pathways) and cell-to-cell contact.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Disease Free

SUBMITTER: Julia Cunha

LAB HEAD: Nathalie Cella

PROVIDER: PXD023224 | Pride | 2021-09-09

REPOSITORIES: Pride

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PI3K-AKT, JAK2-STAT3 pathways and cell-cell contact regulate maspin subcellular localization.

Longhi M T MT Silva L E LE Pereira M M Magalhães M M Reina J J Vitorino F N L FNL Gumbiner B M BM da Cunha J P C JPC Cella N N

Cell communication and signaling : CCS 20210814 1

<h4>Background</h4>Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies indicate that nuclear localization is essential for maspin tumor suppression activity. We have previously shown that the EGFR activation leads to maspin nuclear localization in MCF-10A cells. The present study investigated which EGFR downstream signaling molecules are involved ...[more]

PMID: 34391444

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Project description:Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma. Leucine to arginine substitution at amino acid position 858 (L858R) accounts for around 50% of EGFR-tyrosine kinase inhibitor (TKI) sensitizing mutations. A second site mutation in the gatekeeper residue (T790M) accounts for around 60% of acquired resistance to EGFR-TKIs. We sought to identify the immediate direct and indirect targets of these mutant EGFRs in lung adenocarcinoma and their modulation by erlotinib, the first generation, and most widely used EGFR-directed TKI. We undertook stable isotope labeling of amino acids in cell culture (SILAC), phosphopeptide enrichment, and quantitative mass spectrometry to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring L858R or L858R/T790M mutations and their modulation by erlotinib inhibition. Phosphorylation at the majority of phosphosites identified exhibited no change upon either EGF stimulation or erlotinib inhibition. Only around 7% of phosphosites identified and quantified showed increased phosphorylation upon EGF stimulation in either cell line. However, while phosphorylation at 61% of these phosphosites decreased upon erlotinib inhibition in the TKI sensitive H3255 cells, only 24% of such sites exhibited decreased phosphorylation upon erlotinib inhibition in the TKI resistant H1975 cells. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not the resistant cells include EGFR, Insulin receptor, HGF, MAPK, mTOR, p70S6K and JAK/STAT signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutive phosphorylated in these lung adenocarcinoma cells, but phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinase families, AGC, CAMK and STE were significantly enriched and those of proline directed kinase families, CMGC and CK were significantly depleted among substrates that exhibit increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma.

Dataset Information

PI3K-AKT pathway and cell-cell contact regulates maspin nuclear localization

Publications

PI3K-AKT, JAK2-STAT3 pathways and cell-cell contact regulate maspin subcellular localization.

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