Gene expression profiling of fast- and slow-growing non-functioning gonadotroph pituitary adenomas

in European Journal of Endocrinology
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Objective

Reliable biomarkers associated with aggressiveness of non-functioning gonadotroph adenomas (GAs) are lacking. As the growth of tumor remnants is highly variable, molecular markers for growth potential prediction are necessary. We hypothesized that fast- and slow-growing GAs present different gene expression profiles and reliable biomarkers for tumor growth potential could be identified, focusing on the specific role of epithelial-mesenchymal transition (EMT).

Design and methods

Eight GAs selected for RNA sequencing were equally divided into fast- and slow-growing group by the tumor volume doubling time (TVDT) median (27.75 months). Data were analyzed by tophat2, cufflinks and cummeRbund pipeline. 40 genes were selected for RT-qPCR validation in 20 GAs based on significance, fold-change and pathway analyses. The effect of silencing MTDH (metadherin) and EMCN (endomucin) on in vitro migration of human adenoma cells was evaluated.

Results

350 genes were significantly differentially expressed (282 genes upregulated and 68 downregulated in the fast group, P-adjusted <0.05). Among 40 selected genes, 11 showed associations with TVDT (−0.669<R<−0.46, P < 0.05). These were PCDH18, UNC5D, EMCN, MYO1B, GPM6A and six EMT-related genes (SPAG9, SKIL, MTDH, HOOK1, CNOT6L and PRKACB). MTDH, but not EMCN, demonstrated involvement in cell migration and association with EMT markers.

Conclusions

Fast- and slow-growing GAs present different gene expression profiles, and genes related to EMT have higher expression in fast-growing tumors. In addition to MTDH, identified as an important contributor to aggressiveness, the other genes might represent markers for tumor growth potential and possible targets for drug therapy.

 

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European Society of Endocrinology

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    Significantly differentially expressed genes between fast and slow non-functioning gonadotroph adenomas by RNA-seq. (A) Heatmap showing gene expression profiles clustered across samples and genes. Gene expression levels for each adenoma (n = 8, S = slow, F = fast) are presented in horizontal rows with colors indicating upregulated (red) or downregulated (blue) genes. A total of 350 differentially expressed genes, 282 genes upregulated and 68 genes downregulated in the fast group were found (P-adjusted <0.05). Each column represents a single gene. (B) Distribution of fold-change for selected genes (n = 40) in fast vs slow non-functioning gonadotroph adenomas. Positive fold-change indicates higher gene expression in the fast group. Negative fold-change indicates higher gene expression in the slow group. (C) Epithelial-mesenchymal transition (EMT) and cancer involvement of selected genes (n = 40). The symbol ‘x’ marks the EMT- and cancer-related genes respectively.

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    RT-qPCR validation of 40 selected genes. (A) Associations between relative gene expression of the selected genes (n = 40) and initial TVDT, growth models (exponential, linear and logistic), pre- and post- operative volume, and pre- and post- operative invasiveness in the non-functioning gonadotroph adenomas (n = 20). Invasiveness was measured according to the Knosp–Steiner criteria (45), and also by the tumor’s superior expansion (0 = no superior growth, 1 = upward convex bulging of the sella roof, 2 = touching the optic chiasm, 3 = lifting the optic chiasm, 4 = blockage of interventricular foramina). (B) Scatter plots representing the associations between initial TVDT and significantly associated genes (n = 11) in the non-functioning gonadotroph adenomas (n = 20).

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    Silencing of MTDH and EMCN in primary human adenoma cells. (A) MTDH mRNA levels in scramble control (scramble ctrl) vs silenced MTDH (siMTDH) cells. (B) EMCN mRNA levels in scramble control (scramble ctrl) vs silenced EMCN (siEMCN) cells. (C and D) MTDH protein expression by Western blot in scramble ctrl vs siMTDH. GAPDH was used as reference gene. Data are presented as mean ± s.e.m.; n = 3 patients, 3–4 technical replicates/patient; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

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    Wound-healing assay. (A) Wound-healing assay on silenced MTDH (siMTDH) cells. The pictures (cells are from p.83) illustrate siMTDH cells migrating at a slower rate than scramble control (scramble ctrl) cells. A significant difference was observed after 6 and 31 h (P = 0.03 and 0.021) in patient 73 (p73), after 9, 12 and 24 h (P = 0.013, 0.008 and 0.048) in patient 83 (p83) and after 12, 15, 24, 27 and 29 h (P = 0.04, 0.029, 0.025, 0.019 and 0.019) in patient 86 (p86). AUC shows a significant difference between siMTDH cells and scramble ctrl cells. (B) Wound-healing assay on silenced EMCN (siEMCN) cells. The pictures show siEMCN cells migrating at the same rate as scramble ctrl cells. The only significant difference in cell migration for silenced EMCN (siEMCN) cells was recorded after 6 h (P = 0.014) in p73. No significant difference was observed between siEMCN cells and scramble ctrl cells when calculating AUC. Data are presented as mean ± s.e.m.; n = 3 patients, 4 technical replicates/patient; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; AUC, area under the curve. Scale bar, 500 µm.

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    Epithelial-mesenchymal transition genes. mRNA levels of CDH1 (E-cadherin) and CDH2 (N-cadherin) and the ratio of CDH1/CDH2 in silenced MTDH (siMTDH) and scramble control (scramble ctrl) cells. GAPDH was used as reference gene. Data are presented as mean ± s.e.m.; n = 3 patients, 4 technical replicates/patient; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

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