Introduction of Somatic Mutation in MED12 Induces Wnt4/β-Catenin and Disrupts Autophagy in Human Uterine Myometrial Cell

Abstract

Uterine fibroids (UFs) or leiomyoma are frequently associated with somatic mutations in the mediator complex subunit 12 (MED12) gene; however, the function of these mutations in human UF biology is yet to be determined. Herein, we determined the functional role of the most common MED12 somatic mutation in the modulation of oncogenic Wnt4/β-catenin and mammalian target of rapamycin (mTOR) signaling pathways. Using an immortalized human uterine myometrial smooth muscle cell line (UtSM), we constitutively overexpressed either MED12-Wild Type or the most common MED12 somatic mutation (c.131G>A), and the effects of this MED12 mutation were compared between these cell lines. This immortalized cell line was used as a model because it expresses wild type MED12 protein and do not possess MED12 somatic mutations. By comparing the effect between MED12-WT and MED12-mutant (mut) stable cell populations, we observed increased levels of protein expression of Wnt4 and β-catenin in MED12-mut cells as compared with MED12-WT cells. MED12-mut cells also expressed increased levels of mTOR protein and oncogenic cyclin D1 which are hallmarks of cell growth and tumorigenicity. This somatic mutation in MED12 showed an effect on cell-cycle progression by induction of S-phase cells. MED12-mut cells also showed inhibition of autophagy as compared with MED12-WT cells. Together, these findings indicate that the MED12 somatic mutation has the potentials for myometrial cell transformation by dysregulating oncogenic Wnt4/β-catenin and its downstream mTOR signaling which might be associated with autophagy abrogation, cell proliferation, and tumorigenicity.

Keywords: Leiomyoma; MED12; Somatic mutations; Uterine fibroid.

Fig. 1

Generation of MED12-WT and MED12-mut stable UtSM cell lines. a Plasmid constructs are well-transfected to mammalian cells. 293T cells were transiently transfected with lipofectamin reagent, and 48 h after transfection GFP expression was monitored using confocal pictures. b Lysates from above transfected cells were used to perform western blots using anti-MED12 antibody and anti-Flag antibody. Anti-β-actin antibody was used as a loading control. c MED12-WT and MED12-mut UtSM cell populations were generated by stable transfections as described in the “Materials and Methods” section. Immunoprecipitation assay using lysates from MED12-WT and MED12-mut stable UtSM cell populations showing similar expression of Flag-Tagged MED12-WT and MED12-mut proteins. Similar levels of IgG indicate the equal precipitation. d Equal amounts of protein lysates from MED12-WT and MED12-mut UtSM cell populations were analyzed for endogenous protein expression using anti-MED12 antibody. Similar levels of endogenous MED12 protein were detected in both cell populations. GFP, green fluorescence protein; IP immunoprecipitation; WB Western blot

Fig. 2

Ectopic expression of a MED12-mut Flag-Tagged protein induced Wnt4/β-catenin and promotes cell-cycle progression. a Western blot analyses using lysates from above stable UtSM cell populations showing induced expression of Wnt4 and β-catenin in MED12-mut stable cell populations as compared to MED12-WT stable cell populations. b Cytoplasmic (Cy) and nuclear (Nu) fractions were isolated from cultured MED12-WT and MED12-mut UtSM stable cell populations, and then used for western blot analyses. PARP (nuclear) and RhoGDI (cytoplasmic) western blots are showing the purity of the separation. c FACScan analyses are indicating higher percentage of S-phase cells in MED12-mut stable cell populations as compared with MED12-WT stable cell populations. *P < 0.05 as compared with corresponding control

Fig. 3

Ectopic expression of the MED12-mut Flag-tagged protein in UtSM stable clone induced Wnt4/β-catenin signaling and oncogenic Cyclin D1. Stable clones were generated from MED12-WT and MED12-mut cells populations. Western blot analyses were performed using lysates from these stable clones showing induced expression of β-catenin, Wnt4, and cyclin D1 in MED12-mut stable clone as compared with MED12-WT stable clone. β-actin was used as loading control

Fig. 4

Ectopic expression of a MED12-mut Flag-Tagged protein in UtSM stable clone induced mTOR signaling. Western blot analyses using lysates from MED12-WT and MED12-mut stable clones showing induced expression of phospho-specific p-mTOR and total mTOR in MED12-mut stable clone as compared with MED12-WT stable clone. β-actin was used as loading control

Fig. 5

Altered expression of autophagy-related proteins in MED12-mut UtSM stable clone. a Total RNA was extracted from MED12-WT and MED12-mut stable clones and real-time PCR analyses of mRNA levels of several ATGs were performed as described in the “Materials and Methods” section. Higher expression of ATG3, ATGD4, ATG10, and ATG12 are showing in MED12-mut stable clone as compared with MED12-WT stable clone. Reduced expressions of ATG7 and ATG16 are showing in MED12-mut stable clone as compared with MED12-WT stable clone. b, c Cell lysates from above stable clones were analyzed for protein expression as indicated by each blot. β-actin was used as loading control. d Quantitation of expression of autophagy-related proteins. The intensity of each protein band in b and c was quantified and then the values were normalized with corresponding β-actin. The normalized data graphs were presented as relative protein expression. *p < 0.05 when compared with corresponding control. e Transmission electron microscopy (TME) was used to examine the autophagy status in MED12-WT and MED12-mut stable clones. TME picture shows the blockade of autophagy in MED12-mut stable clone, while it is unaffected in MED12-WT stable clone. Images were taken at × 200 magnification. Black arrows indicate autophagy vacuoles

Fig. 6

Schematic diagram for blockade of autophagy in stable MED12-mut UtSM clone. MED12 somatic mutation can lead to activation of AKT/mTOR signaling pathway that inhibits autophagy. Alternatively, the activation of AKT can inhibit GSK-3β expression that leads to β-catenin upregulation