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TMEM16A controls EGF-induced calcium signaling implicated in pancreatic cancer prognosis.

ABSTRACT: Pancreatic cancer typically spreads rapidly and has poor survival rates. Here, we report that the calcium-activated chloride channel TMEM16A is a biomarker for pancreatic cancer with a poor prognosis. TMEM16A is up-regulated in 75% of cases of pancreatic cancer and high levels of TMEM16A expression are correlated with low patient survival probability. TMEM16A up-regulation is associated with the ligand-dependent EGFR signaling pathway. In vitro, TMEM16A is required for EGF-induced store-operated calcium entry essential for pancreatic cancer cell migration. TMEM16A also has a profound impact on phosphoproteome remodeling upon EGF stimulation. Moreover, molecular actors identified in this TMEM16A-dependent EGFR-induced calcium signaling pathway form a gene set that makes it possible not only to distinguish neuro-endocrine tumors from other forms of pancreatic cancer, but also to subdivide the latter into three clusters with distinct genetic profiles that could reflect their molecular underpinning.

SUBMITTER: Crottes D

PROVIDER: S-EPMC6600921 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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TMEM16A controls EGF-induced calcium signaling implicated in pancreatic cancer prognosis.

Crottès David D Lin Yu-Hsiu T YT Peters Christian J CJ Gilchrist John M JM Wiita Arun P AP Jan Yuh Nung YN Jan Lily Yeh LY

Proceedings of the National Academy of Sciences of the United States of America 20190610 26

Pancreatic cancer typically spreads rapidly and has poor survival rates. Here, we report that the calcium-activated chloride channel TMEM16A is a biomarker for pancreatic cancer with a poor prognosis. TMEM16A is up-regulated in 75% of cases of pancreatic cancer and high levels of TMEM16A expression are correlated with low patient survival probability. TMEM16A up-regulation is associated with the ligand-dependent EGFR signaling pathway. In vitro, TMEM16A is required for EGF-induced store-operated ...[more]

PMID: 31182586

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Project description:BackgroundPhosphotyrosine kinase (PTK)-mediated phospholipase C-γ1 (PLC-γ1) signaling plays a crucial role in the release of the universal second messenger calcium from intracellular stores, which is mandatory for several cellular processes, including cell migration. However, PLC-γ1 could also be activated in a PTK-independent manner by phospholipase D (PLD)-derived phosphatidic acid (PA). Because both higher PLD expression levels and PLD activity have also been associated with breast cancer cell invasion and migration, we wondered whether there might be a link between PLD and PLC-γ1, which was investigated in this study.MaterialsMDA-MB-468-NEO (EGFR positive) and MDA-MB-468-HER2 (EGFR and HER2 positive) human breast cancer cells were used in this study. The migratory behavior of the cells in the presence of epidermal growth factor (EGF) and the PLD inhibitor 5-fluoro-2-indolyl-des-chlorohalopemide (FIPI) was analyzed using the 3D collagen matrix migration assay. Changes in cytosolic calcium levels in the presence of EGF, FIPI and Sig-1R agonists and antagonists as well as in PLD1 siRNA knockdown cells were determined by flow cytometry. Western blot analyses were performed to determine the basal expression levels and phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44, PLC-γ1 and Sig-1R.ResultsThe EGF-induced migration of MDA-MB-468-NEO and MDA-MB-468-HER2 cells was significantly impaired by FIPI. Likewise, FIPI also significantly abolished EGF-induced calcium release in both cell lines. However, neither the expression levels nor the phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44 and PLC-γ1 were markedly changed by FIPI. Knockdown of PLD1 expression by siRNA also significantly impaired EGF-induced calcium release in both cell lines. Targeting Sig-1R, which interacts with IP3R, with the antagonist BD1047 also abrogated EGF-induced calcium release. However, EGF-induced calcium release was also impaired if cells were treated with the Sig-1R agonists PRE084 and PPBP maleate.ConclusionIn summary, blocking PLD activity with the specific inhibitor FIPI or knocking down PDL1 expression by siRNA significantly impaired EGF-induced calcium release in MDA-MB-468-NEO and MDA-MB-468-HER2 cells, likely indicating a connection between PLD activity and PLC-γ1-mediated calcium signaling. However, how PLD activity interferes with the release of calcium from intracellular stores remains unclear. Video Abstract.

Project description:Transforming growth factor (TGF)-? signaling pathway, may act both as a tumor suppressor and as a tumor promoter in pancreatic cancer, depending on tumor stage and cellular context. TGF-? pathway has been under intensive investigation as a potential therapeutic target in the treatment of cancer. We hypothesized a correlation between TGF-?R2/SMAD4 expression in the tumor, plasma TGF-?1 ligand level, genetic variation in TGF-B pathway and prognosis of pancreatic cancer.We examined TGF-?R2 and SMAD4 protein expression in biopsy or surgical samples from 91 patients with pancreatic ductal adenocarcinoma (PDAC) using immunohistochemistry. Plasma level of TGF-?1 was measured in 644 patients with PDAC using ELISA. Twenty-eight single nucleotide polymorphisms (SNP) of the TGF-?1, TGF-?2, TGF-?3, TGF-?R1, TGF-?R2, and SMAD4 genes were determined in 1636 patients with PDAC using the Sequenom method. Correlation between protein expression in the tumor, plasma TGF-?1 level, and genotypes with overall survival (OS) was evaluated with Cox proportional regression models.The expression level of TGF-?R2 and SMAD4 as an independent marker was not associated with OS. However, patients with both low nuclear staining of TGF-?R2 and high nuclear staining of SMAD4 may have better survival (P = 0.06). The mean and median level of TGF-?1 was 15.44 (SD: 10.99) and 12.61 (interquartile range: 8.31 to 19.04) ng/ml respectively. Patients with advanced disease and in the upper quartile range of TGF-?1 level had significantly reduced survival than those with low levels (P = 0.02). A significant association of SMAD4 SNP rs113545983 with overall survival was observed (P<0.0001).Our data provides valuable baseline information regarding the TGF-? pathway in pancreatic cancer, which can be utilized in targeted therapy clinical trials. High TGF-?1 plasma level, SMAD4 SNP or TGF-?R2/SMAD4 tumor protein expression may suggest a dependence on this pathway in patients with advanced pancreatic cancer.

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TMEM16A controls EGF-induced calcium signaling implicated in pancreatic cancer prognosis.

Publications

TMEM16A controls EGF-induced calcium signaling implicated in pancreatic cancer prognosis.

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