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. 2014 Dec 5;4:7330.
doi: 10.1038/srep07330.

Inositol Polyphosphate-5-Phosphatase F (INPP5F) Inhibits STAT3 Activity and Suppresses Gliomas Tumorigenicity

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Free PMC article

Inositol Polyphosphate-5-Phosphatase F (INPP5F) Inhibits STAT3 Activity and Suppresses Gliomas Tumorigenicity

Hong Sug Kim et al. Sci Rep. .
Free PMC article

Abstract

Glioblastoma (GBM), the most common type of primary malignant brain tumors harboring a subpopulation of stem-like cells (GSCs), is a fast-growing and often fatal tumor. Signal Transducer and Activator of Transcription 3 (STAT3) is one of the major signaling pathways in GSCs maintenance but the molecular mechanisms underlying STAT3 deregulation in GSCs are poorly defined. Here, we demonstrate that Inositol Polyphosphate-5-Phosphatase F (INPP5F), one of the polyphosphoinositide phosphatases, is differentially expressed in GSCs from glioma patients, and is identified as an inhibitor of STAT3 signaling via interaction with STAT3 and inhibition of its phosphorylation. Constitutively expressed INPP5F showed to suppress self-renewal and proliferation potentials of glioblastoma cells and reduced tumorigenicity of glioblastoma. In addition, loss of INPP5F gene in gliomas is significantly correlated with lower overall patient survivals. These findings suggest that INPP5F is a potential tumor suppressor in gliomas via inhibition of STAT3 pathway, and that deregulation of INPP5F may lead to contribution to gliomagenesis.

Figures

Figure 1
Figure 1. INPP5F gene locus is frequently deleted and the expression is deregulated in gliomas.
(A) The box and whisker plots show INPP5F expression in 379 glioblastomas (dark red) and in 10 non-tumor brain samples (blue) analyzed from TCGA dataset. The box indicates the 25% ~ 75% quartile and the bars indicate the upper and lower extreme values within the groups (***: p <0.0001). (B) Median expression intensity of INPP5F (probe 203607) in REMBRANDT dataset represents that INPP5F expression in all types of gliomas is decreased compare to non-tumor brain samples (***: p<0.0001). (C) Copy number heatmap of INPP5F and its neighboring genes from TCGA glioblastomas dataset. The green color in the map indicates deletion and black color represents normal copy numbers. (D) INPP5F is differently expressed in GSCs and established glioblastoma cell lines at the protein level.
Figure 2
Figure 2. INPP5F regulates glioblastoma cell proliferation and self-renewal activities in vitro.
(A) INPP5F overexpression decreased glioblastoma cell proliferation, while INPP5F knockdown increased proliferation activity. (B) Analysis of self-renewal activity represents that INPP5F overexpressing GSCs reduced sphere formation and INPP5F knockdown increased sphere forming activity. (C) INPP5F overexpression shows a decreased colony forming activity. (NS: no significant; * p<0.05; ** p<0.001; compared to control). Error bars represent standard deviation (S.D., experiment performed in triplicates).
Figure 3
Figure 3. INPP5F regulates STAT3 pathway.
(A) Hierarchical clustering heatmap of 59 INPP5F signature genes. The signature genes were derived by contrasting experimental control to the corresponding glioblastoma cell lines with either INPP5F overexpression (GSC1228, GCS923, and U87) or knockdown (GSC211 and GSC827). Green color in heatmap indicates increased expression and red color indicates decreased expression in cells with INPP5F overexpression and knockdown, respectively. The vertical bars represent cell lines. (B) Gene set enrichment analysis of STAT3 signaling pathway in INPP5F overexpressing1228 GSCs, compared with control. The green curve represents running enrichment score profile and the lower panel refers to the gene hits in STAT3 signaling pathway along the rank in ordered expression datasets. The normalized enrichment score for this analysis is -1.97 and the p-value is 0.015. (C) STAT3 activity analysis assay. Nuclear fractions were used for detection of STAT3 activity and observed in wavelength of 450 mn. Data show that the STAT3 activation was inhibited by INPP5F overexpression but enhanced by INPP5F knockdown as pair comparison, (NS: no significant; * p<0.05; ** p<0.001; compared to control). Error bars represent standard deviation (S.D., experiment performed in triplicates).
Figure 4
Figure 4. Immunoprecipitation (IP) analysis for INPP5F interaction with STAT3.
(A) and (B) INPP5F overexpressing cells were lysed and immunoprecipitated with anti-V5 for INPP5F and anti-STAT3. Immunoblot analysis represents that INPP5F interacts with STAT3 in both cell lines with overexpressed INPP5F. (C) IP was performed with anti-V5 for INPP5F and immunoblot analysis represents that dominant negative STAT3 (Y705F) interacts with INPP5F in 293T cells. (D) Schematic diagrams of the structural domains and deletion mutants of STAT3. (E) Serial mutants of STAT3 and INPP5F were co-transfected in 293T cells and immuneprecipitated with anti-V5 for INPP5F. Immunoblot analysis represents that INPP5F interacted with full-length and deleted N-terminal domain of STAT3. Anti-GFP is used for STAT3 (Y705F) and its serial mutants, and anti-V5 for INPP5F.
Figure 5
Figure 5. INPP5F inhibits STAT3 phosphorylation and nuclear translocation.
(A) Immunoblot analysis shows that INPP5F overexpression in glioblastoma cells (GSC923, GSC1228, and U87) inhibits phosphorylation of STAT3 (Y705) and INPP5F knockdown (GSC211 and GSC827) enhances its phosphorylation. (B) Immunoblot analysis of nuclear [N] and cytosol [C] fractions reveals that overexpression of INPP5F inhibits STAT3 nuclear translocation while INPP5F knockdown enhances the nuclear translocation of STAT3. (C) Control and INPP5F overexpressing GSC923 cells were treated with IL-6 (50 ng/ml) for 5 and 10 min, and immunoblot was performed with anti-p-STAT3 (Y705). Immunoblot analysis shows that INPP5F inhibits IL-6 induced phosphorylation of STAT3. (D) Confocal microscopy analysis verified that overexpression of INPP5F decreases STAT3 phosphorylation and nuclear translocation compared to control.
Figure 6
Figure 6. Correlation of INPP5F expression with gliomagenesis and survival.
(A) INPP5F expression is correlated with GBM patient survival when glioma specimens are segregated in terms of copy number status using REMBRANDT database. Deletion of INPP5F correlated with poor patient survival, compared to that with unaltered INPP5F. (B) INPP5F overexpressing U87 cells intracranial injected mice show suppressed tumorigenesis compared to control U87 injected mice. Kaplan–Meier survival curves show a median survival time of 85.5 days for mice injected with INPP5F overexpressing U87 cells and 39.5 days for controls (log rank test, *p<0.05). (C) Intracranial tumor histopathological examination. Tumor sections were stained with hematoxylin and eosin (H&E) and examined for evidence of tumor mass at low and high magnification (upper and lower panels, respectively, of each pair). Compared to controls, mice injected with INPP5F overexpressing U87 cells show decreased tumor size and aggressiveness.

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