Increased expression of the microRNA 106b~25 cluster and its host gene MCM7 in corticotroph pituitary adenomas is associated with tumor invasion and Crooke's cell morphology

Abstract

Purpose: MCM7 (minichromosome maintenance complex component 7), a DNA replication licensing factor, is a host gene for the oncogenic miR-106b~25 cluster. It has been recently revealed as a relevant prognostic biomarker in a variety of cancers, including pituitary adenomas. The purpose of this study was to assess whether miR-106b~25 and MCM7 levels correlate with tumor invasiveness in a cohort of ACTH-immunopositive adenomas.

Methods: Tissue samples were obtained intraoperatively from 25 patients with pituitary adenoma. Tumor invasiveness was assessed according to the Knosp grading scale. MCM7, Ki-67 and TP53 levels were assessed by immunohistochemical staining, while the expression of miR-106b-5p, miR-93-5p, miR-93-3p and miR-25-3p were measured using quantitative real-time PCR performed on RNA isolated from FFPE tissues.

Results: We have found a significant increase in MCM7 and Ki-67 labeling indices in invasive ACTHomas. Moreover, MCM7 was ubiquitously overexpressed in Crooke's cell adenomas. The expression of miR-93-5p was significantly elevated in invasive compared to noninvasive tumors. In addition, all four microRNAs from the miR-106b~25 cluster displayed marked upregulation in Crooke's cell adenomas. Remarkably, MCM7 and miR-106b-5p both strongly correlated with Knosp grade. A combination of MCM7 LI and miR-106b~25 cluster expression was able to accurately differentiate invasive from noninvasive tumors and had a significant discriminatory ability to predict postoperative tumor recurrence/progression.

Conclusions: miR-106b~25 and its host gene MCM7 are potential novel biomarkers for invasive ACTH-immunopositive pituitary adenomas. Additionally, they are both significantly upregulated in rare Crooke's cell adenomas and might therefore contribute to their aggressive phenotype.

Keywords: ACTHoma; Crooke’s cell adenoma; MCM7; MiR-106b~25 cluster; MicroRNA; Pituitary adenoma.

Fig. 1

Pituitary corticotroph adenomas. a Ultrastructural features of densely granulated corticotroph adenoma: well-developed organelles, numerous, variable in shape, and electron dense secretory granules. Original magnification ×7400. b Electron microscopy of sparsely granulated corticotroph adenoma: small and scanty secretory granules. Original magnification ×9700. c H&E staining of Crooke’s cell adenoma (×200). d Ultrastructural features of Crooke’s cell adenoma: excessive accumulation of perinuclear cytokeratin filaments. Original magnification ×9700

Fig. 2

A schematic representation of the polycistronic miR-106b~25 cluster and its host gene MCM7, located on chromosome 7q22

Fig. 3

Immunohistochemical analysis of MCM7 expression in ACTH-producing pituitary adenoma samples. Presented are representative cases of MCM7-stained Crooke’s cell (a—2, b—3b), invasive (c—6, d—7a) and noninvasive (e—20, f—22) PAs. MCM7 (g) and Ki-67 (h) LIs are presented as % of positively stained cells in examined PAs. Columns represent means ± SEM. The subgroups were compared using the two-sided Mann–Whitney test: *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

The elements of the miR-106b~25 cluster are coexpressed and upregulated in Crooke’s cell and invasive adenomas. a–d The expression of miR-106b~25 cluster elements in three subgroups of PAs was compared using the two-sided Mann–Whitney test: *p < 0.05, **p < 0.01, ***p < 0.001. e The table presents Pearson correlation coefficients between the expression levels of microRNAs studied. All miRNAs are significantly coexpressed in PAs (p < 0.0001 for all coefficients)

Fig. 5

MCM7 and miR-106b-5p are coexpressed in ACTHomas. a–d The correlation was calculated using Pearson’s coefficient

Fig. 6

The expression of miR-106b-5p and MCM7 LI correlates with tumor’s Knosp grade. Scatter plots showing the relationship between tumor’s Knosp grade and miR-106b~25 expression (a–d) as well as MCM7 LI (e). Correlation was characterized by calculating Spearman’s rank coefficient

Fig. 7

MCM7 LI in combination with the expression of miR-106b~25 is a specific and sensitive test discriminating invasive from noninvasive ACTHomas. a In order to establish the role of the IHC biomarkers and microRNAs studied, we analyzed the area under the receiver operating characteristic (AUC ROC). b A graphical representation of the three top AUC values

Fig. 8

Performance of MCM7 LI, miR-106b~25 expression and Ki67 LI in identifying patients with unfavorable prognosis. a A summary of AUC ROC calculated for progression/recurrence and post-operative residual tumor accompanied by a graphical representation of those values (b, c). d The expression of MCM7 in residual tumors that progressed or remained stable calculated using the two-sided Mann–Whitney test: *p < 0.05