Functional screen analysis reveals miR-26b and miR-128 as central regulators of pituitary somatomammotrophic tumor growth through activation of the PTEN-AKT pathway

Abstract

MicroRNAs (miRNAs) have been involved in the pathogenesis of different types of cancer; however, their function in pituitary tumorigenesis remains poorly understood. Cyclic-AMP-dependent protein kinase-defective pituitaries occasionally form aggressive growth-hormone (GH)-producing pituitary tumors in the background of hyperplasia caused by haploinsufficiency of the protein kinase's main regulatory subunit, PRKAR1A. The molecular basis for this development remains unknown. We have identified a 17-miRNA signature of pituitary tumors formed in the background of hyperplasia (caused in half of the cases by PRKAR1A-mutations). We selected two miRNAs on the basis of their functional screen analysis: inhibition of miR-26b expression and upregulation of miR-128 suppressed the colony formation ability and invasiveness of pituitary tumor cells. Furthermore, we identified that miR-26b and miR-128 affected pituitary tumor cell behavior through regulation of their direct targets, PTEN and BMI1, respectively. In addition, we found that miR-128 through BMI1 direct binding on the PTEN promoter affected PTEN expression levels and AKT activity in the pituitary tumor cells. Our in vivo data revealed that inhibition of miR-26b and overexpression of miR-128 could suppress pituitary GH3 tumor growth in xenografts. Taken together, we have identified a miRNA signature for GH-producing pituitary tumors and found that miR-26b and miR-128 regulate the activity of the PTEN-AKT pathway in these tumors. This is the first suggestion of the possible involvement of miRNAs regulating the PTEN-AKT pathway in GH-producing pituitary tumor formation in the context of hyperplasia or due to germline PRKAR1A defects. MiR-26b suppression and miR-128 upregulation could have therapeutic potential in GH-producing pituitary tumor patients.

Figure 1

MicroRNA signature of GH-producing pituitary tumors. (a) Heatmap representation of differentially expressed microRNAs identified by microRNA array analysis between normal and cancer (patients 1–7) tissues. (b) MiR-26b, miR-212, let-7a3 and miR-128 expression levels assessed by real-time PCR analysis in 5 normal and 7 cancer pituitary tissues. The experiment has been performed in triplicate and data are shown as mean ± SD.

Figure 2

MiR-26b and miR-128 regulate the colony formation ability and invasiveness of pituitary tumor cells. (a) Number of colonies (mean ± SD) of AtT-20 pituitary cells untreated or treated with 50nM microRNAs (miR-125b, miR-141, miR-144, miR-164, miR-145, miR-143, miR-15b, miR-16, miR-186, let-7b, let-7a3, miR-128) or anti-sense microRNAs (as-miR-26b, as-miR-26a, miR-128, as-miR-212, as-miR-107, as-miR-103) for 48h. (b) Schematic of microRNA library screen in AtT-20 cells in order to assess which microRNAs affect their invasiveness. (c) Number of invading cells (mean ± SD) of AtT-20 pituitary cells untreated or treated with 50nM microRNAs (miR-125b, miR-141, miR-144, miR-164, miR-145, miR-143, miR-15b, miR-16, miR-186, let-7b, let-7a3, miR-128) or anti-sense microRNAs (as-miR-26b, as-miR-26a, miR-128, as-miR-212, as-miR-107, as-miR-103).

Figure 3

MiR-26b regulates PTEN expression and miR-128 regulates BMI1 expression in pituitary cancers. (a) Heatmap representation of PTGS2, PTEN, HMGA1, MAPK14, PLK2 and BMI1 mRNA expression levels in 7 pituitary tumors assessed by real-time PCR analysis. (b) Sequence complementarity between miR-26b and the 3’UTR of PTEN gene and between miR-128 and the 3’UTR of BMI1 gene. (c) PTEN 3’UTR luciferase activity (mean ± SD) and (d) mRNA expression levels (mean ± SD) in untreated or miR negative control (miR-NC) and miR-26b treated HEK293 cells for 24h.

Figure 4

MiR-26b and miR-128 control the tumorigenicity and invasiveness of pituitary tumors cell through regulation of PTEN and BMI1, respectively. (a) Number of colonies (mean ± SD) and (b) invading AtT-20 cells untreated or treated with 50nM antisense-microRNA negative control (as-miR-NC), antisense-microRNA-26b (as-miR-26b), antisense-microRNA-128 (asmiR-128), siRNA negative control (siRNA NC) and siRNA against PTEN (siRNA-PTEN).

Figure 5

MiR-26 and miR-128 regulate the PTEN-AKT pathway in AtT-20 pituitary cells. (a) Number of colonies and (b) invading AtT-20 cells, untreated or treated with 50nM miR-NC, asmiR-NC, as-miR-26b and miR-128. (c) Fold enrichment of BMI1 in the promoter area of PTEN in AtT-20 cells treated with 50NM as-miR-NC or as-miR-128, assessed by chromatin immunoprecipitation followed by real-time PCR analysis. (d) PTEN mRNA expression levels (mean ± SD) assessed by real-time PCR analysis and E, AKT phosphorylation levels (S473) in AtT-20 cells treated for 48h with 50nM as-miR-NC, as-miR-128, miR-26b and their combinations. (f) Number of colonies and invading AtT-20 cells, untreated or treated with 50nM as-miR-26b and miR-128 or combination of as-miR-26b, miR-128 and siRNA NC or combination of as-miR-26b, miR-128 and siRNA-PTEN. The experiments have been performed in triplicate and data are shown as mean ± SD.

Figure 6

MicroRNA-gene networks regulating pituitary oncogenesis. (a) Correlation (r represents the correlation coefficient) in the expression levels of miR-26b, miR-128, PTEN and BMI1 in human pituitary tumors. (b) Schematic of miR-26b and miR-128 signaling pathway in pituitary oncogenesis.