CPVL promotes glioma progression via STAT1 pathway inhibition through interactions with the BTK/p300 axis
Hui Yang, … , Xiuming Liang, Kun Lv
Hui Yang, … , Xiuming Liang, Kun Lv
Published November 16, 2021
Citation Information: JCI Insight. 2021;6(24):e146362. https://doi.org/10.1172/jci.insight.146362.
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Research Article Oncology

CPVL promotes glioma progression via STAT1 pathway inhibition through interactions with the BTK/p300 axis

Abstract

CPVL (carboxypeptidase, vitellogenic-like) is a serine carboxypeptidase that was first characterized in human macrophages. However, the function of CPVL remains unclear in a variety of tumors. The quantitative PCR (qPCR), Western blotting, and IHC assays were utilized to measure the CPVL expression. CPVL was significantly upregulated in glioma cells and tissues compared with normal cells and tissues, respectively. Moreover, high CPVL expression was correlated with advanced clinical grade and poor prognosis. Silencing of CPVL promoted glioma cell apoptosis, and it inhibited cell proliferation and tumorigenicity in vitro and in vivo. Ingenuity Pathway Analysis (IPA) demonstrated that CPVL silencing activated the IFN-γ/STAT1 signaling pathway, thereby inducing glioma cell apoptosis. Mechanistically, immunopurification, mass spectrometry, IP, and glutathione S-transferase (GST) pull-down experiments elucidated that CPVL physically interacts with Bruton’s tyrosine kinase (BTK) and downregulates the STAT1 phosphorylation through promoting p300-mediated STAT1 acetylation. Our findings reveal the crucial role of CPVL in promoting the progression of glioma through suppressing STAT1 phosphorylation. CPVL might serve as a potential prognostic biomarker and therapeutic target for the treatment of glioma.

Authors

Hui Yang, Xiaocen Liu, Xiaolong Zhu, Xueqin Li, Lan Jiang, Min Zhong, Mengying Zhang, Tianbing Chen, Mingzhe Ma, Xiuming Liang, Kun Lv

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Figure 7

CPVL physically interacts with BTK to regulate the STAT1 phosphorylation through p300-mediated STAT1 acetylation.

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CPVL physically interacts with BTK to regulate the STAT1 phosphorylation...
(A) Immunoaffinity purification of CPVL-containing protein complex. Cellular extracts from U251 cells stably expressing FLAG vector or FLAG-CPVL were immunopurified with anti-FLAG affinity columns and eluted with FLAG peptide. These eluates were resolved by SDS-PAGE and were silver stained. (B) IP of whole-cell lysates from U251 cells followed by IB with antibodies against the indicated proteins. (C) GST pull-down assays with GST-fused CPVL and in vitro transcribed/translated TARA, BTK, MICA, PQBP1, or VAPA as indicated. (D) GST pull-down assays with the indicated GST-fused proteins and in vitro transcribed/translated CPVL. (E and F) U251 cells were transfected with a control shRNA or CPVL shRNA. The mRNA level of BTK and the protein levels of CPVL, BTK, and p-BTK were measured. (G) U251 cells were treated with control cDNA or BTK DN cDNA, and the protein levels of p-BTK, BTK, p300, PY20 (IP: p300), Ac-Lys (IP: STAT1), p-STAT1, and STAT1 were measured by Western blotting. (H) Rescue experiments were used to investigate whether the biological function of CPVL was mediated by regulating phosphorylation of BTK. U251 cells were transfected with CPVL shRNA or CPVL shRNA plus BTK cDNA compared with the control cells, and the protein levels of CPVL, p-BTK, BTK, p300, PY20 (IP: p300), Ac-Lys (IP: STAT1), p-STAT1, and STAT1 were measured by Western blotting. (I) Rescue experiments were used to investigate the STAT1 phosphorylation in CPVL-silenced U251 cells treated with acetylation activator, acetyl resveratrol, compared with the control cells, and the protein levels of CPVL, STAT1, Ac-Lys (IP: STAT1), and p-STAT1 were measured by Western blotting. All experiments were repeated 3 times. β-Actin was used as a loading control. Bar graph data are presented as mean ± SD. Two-tailed Student’s t test analyses were performed.