Fig. 7

TMEM52B suppression reduces E-cadherin stability and promotes extracellular shedding of E-cadherin, resulting in EGFR phosphorylation. (A) HCT-15 cells transfected with shRNA specific to TMEM52B for 48 h were immunofluorescent stained for E-cadherin (red) and nuclei (blue). Differential interference contrast (DIC) images are also shown. Scale bar, 10 μm. The level of E-cadherin-staining was calculated based on the ratio of the E-cadherin-positive area to the total observation area using ImageJ software. At least 7 random fields were analyzed. (B) Cells transfected with shRNA specific to TMEM52B for 42 h were treated with MG132 (2 μM) for 6 h before lysis for immunoblot analysis. (C) Co-immunoprecipitation analysis of the interaction between TMEM52B and E-cadherin in HEK293E cells co-transfected with myc-tagged TMEM52B and E-cadherin. Whole-cell lysates were immunoprecipitated with anti-myc and analyzed by immunoblotting with anti-myc or anti-E-cadherin. (D, E) Cells were transfected with shRNA specific to TMEMT52B for 48 h. (D) Transfected cells were lysed for immunoblotting. (E) A cytosolic fraction (denoted by C), nuclear fraction (N), and plasma membrane-enriched fraction (M) were prepared from transfected HCT-15 cells for immunoblot analysis of β-catenin. GAPDH, histone H3, and EGFR were used as internal controls for the cytosolic, nuclear, and plasma membrane fractions, respectively. Densitometric quantification was performed on the immunoblots using GAPDH or a fractional internal control as a loading control. (F) Transcriptional activity of β-catenin was analyzed using the TOP/FOP reporter system. HCT-15 cells were co-transfected with shRNA specific to TMEM52B and reporter plasmid for 48 h. Firefly luciferase activity was normalized against the Renilla luciferase activity and fold increases in TOPFlash activity compared to FOPFlash activity were calculated. (G) Cells were co-transfected with shRNA specific to TMEM52B and an E-cadherin promoter (− 308/+ 41) reporter plasmid for 48 h. E-cadherin promoter reporter activity was then measured. All determinations were performed in three independent experiments. Values represent mean ± SD. *P < 0.05. (H) HCT-15 cells were transfected with TMEM52B-specific shRNA for 48 h and the conditioned media from transfected cells collected for an additional 48 h. Lysates (intact E-cadherin) from transfected cells, and conditioned media (soluble E-cadherin; denoted as sE-cad) from cells were analyzed by immunoblotting. (I) Cells were serum-starved for 24 h and then treated with recombinant extracellular E-cadherin protein fused with Fc for 8 h. Cells were also treated with 25 μg/ml recombinant Fc protein as a negative control. Lysates were prepared and analyzed by immunoblotting. (J) A schematic representation illustrating the pathways for TMEM52B action in human cancer cells. TMEM52B suppression promoted activation and internalization of EGFR with enhanced MAPK and AKT signaling activity, leading to enhanced cell survival and invasion. In addition, TMEM52B suppression reduced E-cadherin stability, likely due to a reduced association between it and E-cadherin, resulting in enhanced β-catenin transcriptional activity. TMEM52B suppression-mediated generation of a soluble E-cadherin fragment, at least partially, contributed to EGFR activation. EE, early endosome. Nucleus was not distinguished. Dashed arrows indicate translocation