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, 42 (8), 604-616

Phosphoserine Phosphatase Promotes Lung Cancer Progression Through the Dephosphorylation of IRS-1 and a Noncanonical L-Serine-Independent Pathway

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Phosphoserine Phosphatase Promotes Lung Cancer Progression Through the Dephosphorylation of IRS-1 and a Noncanonical L-Serine-Independent Pathway

Seong-Min Park et al. Mol Cells.

Abstract

Phosphoserine phosphatase (PSPH) is one of the key enzymes of the L-serine synthesis pathway. PSPH is reported to affect the progression and survival of several cancers in an L-serine synthesis-independent manner, but the mechanism remains elusive. We demonstrate that PSPH promotes lung cancer progression through a noncanonical L-serine-independent pathway. PSPH was significantly associated with the prognosis of lung cancer patients and regulated the invasion and colony formation of lung cancer cells. Interestingly, L-serine had no effect on the altered invasion and colony formation by PSPH. Upon measuring the phosphatase activity of PSPH on a serine-phosphorylated peptide, we found that PSPH dephosphorylated phospho-serine in peptide sequences. To identify the target proteins of PSPH, we analyzed the protein phosphorylation profile and the PSPH-interacting protein profile using proteomic analyses and found one putative target protein, IRS-1. Immunoprecipitation and immunoblot assays validated a specific interaction between PSPH and IRS1 and the dephosphorylation of phospho-IRS-1 by PSPH in lung cancer cells. We suggest that the specific interaction and dephosphorylation activity of PSPH have novel therapeutic potential for lung cancer treatment, while the metabolic activity of PSPH, as a therapeutic target, is controversial.

Keywords: IRS-1; L-serine independent pathway; lung cancer; phosphoserine phosphatase.

Conflict of interest statement

Disclosure

The authors have no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1. Promotion of lung cancer progression by PSPH
(A) Prognoses of two groups of lung cancer patients classified by PSPH expression. Red, high expression group; Blue, low expression group. (B) Alteration frequency of PSPH in lung cancer patients (TCGA, cBioPortal, Oncoprint). (C) Correlation between CNA and PSPH expression in lung cancer patients (TCGA).
Fig. 2
Fig. 2. Promotion of lung cancer cell invasion and colony formation by PSPH in vitro
After PSPH knockdown: RT-qPCR (A), invasion assay (B), and colony-forming assay (C). After ectopic PSPH overexpression: immunoblot assay (D), invasion assay (E), and colony-forming assay (F). Data are representative of three independent experiments. The error bars represent the standard error of the mean.
Fig. 3
Fig. 3. Regulation of protein phosphorylation by PSPH
(A) Phosphatase activity assay on phosphopeptides using PSPH IP samples. Data are representative of three independent experiments. The error bars represent the standard error of the mean. (B) Differentially (serine-) phosphorylated proteins by PSPH knockdown (high-throughput ELISA-based phosphoantibody array). (C) LC-MS/MS analysis with PSPH IP samples. (D) Venn analysis with the LC-MS/MS and phosphoantibody array data.
Fig. 4
Fig. 4. Regulation of IRS-1 phosphorylation and downstream signals by PSPH
(A) Validation of the specific interaction between PSPH and IRS-1 proteins. (B) Validation of IRS-1 dephosphorylation by PSPH overexpression (immunoblot assay). (C) Validation of IRS-1 dephosphorylation by wild type (WT) and mutant (D20A) PSPH (immunostaining). Red, phospho-IRS-1 (Ser794); Blue, DAPI. (D) Regulation of downstream signals by WT and mutant (D20A) PSPH. (E) Results of the RT-qPCR assay for the known downstream target genes of the Akt signaling pathway (CDK2: P = 4.0 × 10−5, HRAS: P = 7.0 × 10−5, MAPKAP1: P = 8.0 × 10−4, SLC2A1: P = 1.4 × 10−6; t-test). (F) The correlation between PSPH and Akt target genes. (G) The results of invasion assays with rapamycin treatment after ectopic PSPH overexpression (NCI-H1299: P = 5.8 × 10−5, A549: P = 7.4 × 10−5; t-test). Data are representative of three independent experiments. The error bars represent the standard error of the mean. ***P < 0.001.
Fig. 5
Fig. 5. Regulation of lung cancer by PSPH in vivo
Mouse xenograft after PSPH knockdown: tumor volume (A), tumor weight (B), and tumor size (the error bars represent the standard deviation of the mean) (C). (D) Result of the IHC staining with tumor tissues of mouse xenograft model. (E) Result of the IHC staining with lung cancer tissue microarray.

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