Reduced menin expression impairs rapamycin effects as evidenced by an increase in mTORC2 signaling and cell migration

Abstract

Background: Mammalian target of rapamycin (mTOR) is a master regulator of various cellular responses by forming two functional complexes, mTORC1 and mTORC2. mTOR signaling is frequently dysregulated in pancreatic neuroendocrine tumors (PNETs). mTOR inhibitors have been used in attempts to treat these lesions, and prolonged progression free survival has been recorded. If this holds true also for the multiple endocrine neoplasia type 1 (MEN1) associated PNETs is yet unclear. We investigated the relationship between expression of the MEN1 protein menin and mTOR signaling in the presence or absence of the mTOR inhibitor rapamycin.

Methods: In addition to use of menin wild type and menin-null mouse embryonic fibroblasts (MEFs), menin was silenced by siRNA in pancreatic neuroendocrine tumor cell line BON-1. Panels of protein phosphorylation, as activation markers downstream of PI3k-mTOR-Akt pathways, as well as menin expression were evaluated by immunoblotting. The impact of menin expression in the presence and absence of rapamycin was determinate upon Wound healing, migration and proliferation in MEFs and BON1 cells.

Results: PDGF-BB markedly increased phosphorylation of mTORC2 substrate Akt, at serine 473 (S473) and threonine 450 (T450) in menin^-/- MEFs but did not alter phosphorylation of mTORC1 substrates ribosomal protein S6 or eIF4B. Acute rapamycin treatment by mTORC1-S6 inhibition caused a greater enhancement of Akt phosphorylation on S473 in menin^-/- cells as compared to menin^+/+ MEFs (116% vs 38%). Chronic rapamycin treatment, which inhibits both mTORC1and 2, reduced Akt phosphorylation of S473 to a lesser extent in menin^-/- MEFs than menin^+/+ MEFs (25% vs 75%). Silencing of menin expression in human PNET cell line (BON1) also enhanced Akt phosphorylation at S473, but not activation of mTORC1. Interestingly, silencing menin in BON1 cells elevated S473 phosphorylation of Akt in both acute and chronic treatments with rapamycin. Finally, we show that the inhibitory effect of rapamycin on serum mediated wound healing and cell migration is impaired in menin^-/- MEFs, as well as in menin-silenced BON1 cells.

Conclusions: Menin is involved in regulatory mechanism between the two mTOR complexes, and its reduced expression is accompanied with increased mTORC2-Akt signaling, which consequently impairs anti-migratory effect of rapamycin.

Keywords: Akt; MEN1; PI3K; PNET; Rapamycin; mTOR.

Fig. 1

Menin−/− MEFs show enhanced mTORC2, but not mTORC1 activation. Serum-starved menin+/+ and menin−/− MEF cells were cultured in the presence or absence of PDGF-BB (20 ng/ml) at 37 °C for indicated time periods (a), or for 30 min (a & b), with or without pretreatment with the mTOR inhibitor rapamycin (100 nM) for 1 h (b) or 24 h (c). Total cell lysate (TCL) were analyzed by immunoblotting with antibodies specific for menin, Akt phosphorylated on S473 or T450, or total Akt, as well as mTOR, eIF4B and S6 phosphorylation, and the expression of respective total protein. The relative protein phosphorylations were quantified for a representative experiment. Note enhancement of p-Akt levels, but not p-S6, in menin−/− cells (a). Menin−/− cells show enhanced p-Akt after short (1 h) rapamycin treatment (b). The inhibitory effect of prolonged (24 h) rapamycin treatment on p-Akt is menin dependent (c)

Fig. 2

Schematic figure depicting the key roles of menin in mTOR signaling. Initially, PDGF-BB or serum mediated activation of Akt involves PI3K-mTORC2 pathway with menin as negative regulator. Activation of S6 and eIF4B occurs in a PI3K/MAPK dependent manner, but independent of mTORC2-Akt signaling, or menin protein expression. There is a direct negative regulatory interplay between menin protein expression and the rapamycin-mediated activation of mTORC2-Akt

Fig. 3

Absence of Menin enhances wound-healing upon rapamycin treatment. Menin−/− and menin+/+ MEFs were subjected to a cell culture wound migration assay as described in Methods. The initial wound space was approximately 0.6 mm. After serum stimulation of cells, or in the presence of rapamycin or dual combination with wortmanin, the cell movement into the gap was imaged with a digital camera using a Zeiss microscope (a). The width of the open wound was measured with the T scratcher software. The data are presented as calculated percentage of the wound closure after 48 h relative to corresponding control (b). Only in menin+/+ MEFs rapamycin treatment significantly prevented wound healing and cell migration (P < 0.05). Data are presented as mean values (± SEM) in 5 independent experiments. Note that rapamycin significantly decreased wound healing in menin+/+ but not in menin−/− cells

Fig. 4

SiRNA-downregulation of menin in BON1 cells enhances Rapamycin-induced Akt phosphorylation and migration. Control or menin-siRNA treated BON1 cells were pretreated with or without rapamycin (100 nM) for indicated time periods upon starvation (a), PDGF-BB (b), or serum condition (c), as specified. Total cell lysate were analyzed by immunoblotting with antibodies specific for menin, Akt phosphorylated on S473, as well as S6 phosphorylation, and the expression of total protein Akt and S6. The relative protein phosphorylations were quantified for a representative experiment. The migration of BON1 cells was carried out as explained in Methods. The 12 well plate were field with medium containing 10% serum as attractant, 200 × 103 pretreated BON1 cells with either control or menin-siRNA, as indicated, were placed on the insert filters to migrate overnight at 37 °C. The amount of the migrated cells is given as percentage relative of corresponding control (d). Only in control siRNA treated BON1 cells rapamycin treatment significantly reduced cell migration (P < 0.01). Data are presented as mean values (± SEM) in three independent experiments, each performed in triplicate

Fig. 5

In absence of menin an additional treatment with rapamycin reactivates mTORC2 signaling. Menin+/+ and menin−/− MEFs, were serum-starved for 24 h and then stimulated with PDGF-BB (20 ng/ml) for 30 min or as specified, with or without pretreatment with the mTOR inhibitor rapamycin (100 nM) for 24 h + 1 h (a), or in combination with PI3 kinase inhibitor wortmannin (0.2 μm) (b). In menin−/− cells rapamycin retreatment markedly increases mTORC2 signaling (a). Enhanced rapamycin-induced p-Akt occurs downstream of PI3K (b). Menin+/+ MEFs were serum starved and stimulated with PDGF-BB for 30 min after pretreatment with Mek1/2 inhibitor CI-1040, PI3K inhibitor wortmannin (0.2 μm), Ca2+ chelators BAPTA-AM (BA, 10 μM) or EDTA (ED, 2 mM) for 30 min, and rapamycin (100 nM) for 24 h, as indicated (c). Total cell lysate were analyzed by immunoblotting with antibodies specific for menin, Akt phosphorylated on S473 and T450, as well as NDRG1, Rictor, S6 and eIF4B phosphorylation, and the expression of βactin. The relative protein phosphorylations were quantified for a representative experiment