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. 2024 May 23;32(6):1079-1091.
doi: 10.32604/or.2024.046007. eCollection 2024.

GNAS mutations suppress cell invasion by activating MEG3 in growth hormone-secreting pituitary adenoma

Chao Tang  1 , Chunyu Zhong  2 , Junhao Zhu  1 , Feng Yuan  1 , Jin Yang  1 , Yong Xu  3 , Chiyuan Ma  1   3   4   5
Affiliations

GNAS mutations suppress cell invasion by activating MEG3 in growth hormone-secreting pituitary adenoma

Chao Tang et al. Oncol Res. .

Abstract

Approximately 30%-40% of growth hormone-secreting pituitary adenomas (GHPAs) harbor somatic activating mutations in GNAS (α subunit of stimulatory G protein). Mutations in GNAS are associated with clinical features of smaller and less invasive tumors. However, the role of GNAS mutations in the invasiveness of GHPAs is unclear. GNAS mutations were detected in GHPAs using a standard polymerase chain reaction (PCR) sequencing procedure. The expression of mutation-associated maternally expressed gene 3 (MEG3) was evaluated with RT-qPCR. MEG3 was manipulated in GH3 cells using a lentiviral expression system. Cell invasion ability was measured using a Transwell assay, and epithelial-mesenchymal transition (EMT)-associated proteins were quantified by immunofluorescence and western blotting. Finally, a tumor cell xenograft mouse model was used to verify the effect of MEG3 on tumor growth and invasiveness. The invasiveness of GHPAs was significantly decreased in mice with mutated GNAS compared with that in mice with wild-type GNAS. Consistently, the invasiveness of mutant GNAS-expressing GH3 cells decreased. MEG3 is uniquely expressed at high levels in GHPAs harboring mutated GNAS. Accordingly, MEG3 upregulation inhibited tumor cell invasion, and conversely, MEG3 downregulation increased tumor cell invasion. Mechanistically, GNAS mutations inhibit EMT in GHPAs. MEG3 in mutated GNAS cells prevented cell invasion through the inactivation of the Wnt/β-catenin signaling pathway, which was further validated in vivo. Our data suggest that GNAS mutations may suppress cell invasion in GHPAs by regulating EMT through the activation of the MEG3/Wnt/β-catenin signaling pathway.

Keywords: EMT; GHPAs; GNAS mutation; MEG3; β-catenin.

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Conflict of interest statement

The authors declare that they have no conflicts of interest to report regarding the present study.

Figures

Figure 1
Figure 1. GNAS mutations inhibit GH3 cell invasion. A: Wild-type and mutant GNAS were expressed in GH3 cells by transduction with lentiviral vectors: pWPT-GNAS-Q227L, pWPT-GNAS-R201C, and pWPT-GNAS. B: Cell invasion was measured using the Transwell assay (magnification, ×200). C: The levels of MMP-2 and MMP-9 were quantified via western blotting. **p < 0.01, ***p < 0.001 show statistical significance between the two groups as indicated, and “ns” shows no significance. GH3-GNAS-WT: GH3 cells containing wild-type GNAS; GH3-Q227L: GH3 cells expressing the mutant GNAS at Q227L; GH3-R201C: GH3 cells expressing the mutant GNAS at R201C. Data represent the mean ± s.d. of three independent experiments
Figure 2
Figure 2. GNAS mutations upregulate MEG3 expression. A: The expression of MEG3 in GHPA and NFPA was quantified via RT-qPCR analysis. B and C: Correlation between MEG3 expression and GNAS mutations was determined via RT-qPCR. D: Correlation between MEG3 expression and percentage of invasive tumors was analyzed. **p < 0.01, ***p < 0.001 show statistical significances between the two groups as indicated. GHPA: growth hormone–secreting pituitary adenoma; NFPA: non‑functioning pituitary adenoma; GNAS-MUT: growth hormone–secreting pituitary adenoma possessing GNAS mutations; GNAS-WT: growth hormone–secreting pituitary adenoma possessing wild-type GNAS; MEG3-high and MEG3-low: GHPAs were divided into the MEG3 high expression group and low expression group according to the MEG3 expression level
Figure 3
Figure 3. MEG3 inhibits the invasiveness of GHPA cells. A: MEG3 was overexpressed or knocked down in GH3 cells. Relative levels of MEG3 were quantified via immunofluorescence assay and confirmed via RT-qPCR (magnification, ×200). B: Cell invasion was analyzed using the Transwell assay (magnification, ×200). C: Expression levels of MMP-2 and MMP-9 in MEG3-overexpressing cells were measured via western blotting. D: Cell invasion of MEG3 knockdown in GH3 cells expressing mutant GNAS was detected using the Transwell assay (magnification, ×200). E: Expression levels of MMP-2 and MMP-9 of MEG3 knockdown in GH3 cells expressing mutant GNAS were measured via western blotting. *p < 0.05, **p < 0.01, and ***p < 0.001 show the statistical significance between the two groups as indicated, and “ns” shows no significance. MEG3-oe-vt: GH3 cells expressing empty vector of overexpressed MEG3; MEG3-oe: GH3 cells expressing overexpressed MEG3; MEG3-si-vt: GH3 cells containing MEG3 siRNA expressing empty vector; MEG3-si: GH3 cells containing MEG3 siRNA expressing vector
Figure 4
Figure 4. MEG3 inhibits the invasiveness of GHPA with GNAS mutations by inactivating Wnt/β-catenin signaling. A: RNA-Seq was used to examine the gene expression profiles in MEG3-overexpressed GH3 cells compared with vector-only controls. The altered mRNA expression profile was analyzed using Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. B: Reduction of β-catenin mRNA expression in GHPA tumors with the GNAS mutations and GH3 cells with a high level of MEG3 was confirmed via RT-qPCR. C: Relative β-catenin levels were measured via western blotting. D: Significant negative relationships were found between the expression levels of MEG3 and β-catenin. E: β-catenin levels between GNAS-mutant and wild-type tumors were analyzed using IHC (magnification, ×200). *p < 0.05 and **p < 0.01 show the statistical significance between the two groups as indicated, and “ns” shows no significance
Figure 5
Figure 5. MEG3-mediated downregulation of β-catenin inhibits GH3 cell invasion. A: MEG3 was manipulated by expressing or silencing in GH3 cells. The expression of β-catenin was induced by LiCl (20 mM) or repressed by Dkk1 (150 ng/mL) and measured using western blotting. B: Relative cell invasion was quantified by the Transwell assay (magnification, ×200). C: Expression of MMP-2 and MMP-9 was measured using western blotting. *p < 0.05, **p < 0.01 show statistical significance between the two groups as indicated
Figure 6
Figure 6. GNAS mutations inhibit EMT in GHPA tumors. A and B: The expression of EMT-related proteins in GHPA tumor tissues was quantified via immunofluorescence (magnification, ×200). C: Mechanism by which GNAS mutations inhibit the invasiveness of GHPA. *p < 0.05, **p < 0.01, and ***p < 0.001 show statistical significance between the two groups as indicated. GPCR: G protein coupled receptor; Gsα: α subunit of Gs protein; GDP: guanosine diphosphate; GTP: guanosine triphosphate; AC: adenylyl cyclase; cAMP: cyclic adenosine monophosphate; PKA: protein kinase A; CREB: cAMP response element-binding protein; CRE: cAMP response element; EMT: epithelial–mesenchymal transition
Figure 7
Figure 7. MEG3 inhibits GHPA cell invasion in vivo. A: GH3 cells expressing different levels of MEG3 were subcutaneously injected into null mice followed by LiCl treatment. B: Sustained efficiency of MEG3 upregulation was verified by detecting the expression level of MEG3 in the xenograft tumor of each group via RT-qPCR. C, D: Levels of β-catenin, E-cadherin, N-cadherin, MMP-2, and MMP-9 in tumor tissues were quantified via immunohistochemistry (IHC) (magnification, ×200). *p < 0.05, **p < 0.01, and ***p < 0.001 show statistical significance among the three groups as indicated, and “ns” shows no significance
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