p53, miR-34a and EMP1-Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

Abstract

Trefoil factor family peptide 3 (TFF3) is supposed to have tumor suppressive functions in retinoblastoma (RB), but the functional pathway is not completely understood. In the study presented, we investigated the downstream pathway of TFF3 signaling in Y79 RB cells. Results from pG13-luciferase reporter assays and western blot analyses indicate induced p53 activity with an upregulation of miR-34a after TFF3 overexpression. Expression levels of the predicted miR-34a target epithelial membrane protein 1 (EMP1) are reduced after TFF3 overexpression. As revealed by WST-1 assay, BrdU, and DAPI cell counts viability and proliferation of Y79 cells significantly decrease following EMP1 knockdown, while apoptosis levels significantly increase. Opposite effects on Y79 cells' growth could be shown after EMP1 overexpression. Caspase assays showed that EMP1 induced apoptosis after overexpression is at least partially caspase-3/7 dependent. Colony formation and soft agarose assays, testing for anchorage independent growth, revealed that EMP1 overexpressing Y79 cells have a significantly higher ability to form colonies. In in ovo chicken chorioallantoic membrane (CAM) assays inoculated EMP1 overexpressing Y79 cells form significantly larger CAM tumors. Moreover, miR-34a overexpression increases sensitivity of Y79 cells towards RB chemotherapeutics, however, without involvement of EMP1. In summary, the TFF3 signaling pathway in Y79 RB cells involves the activation of p53 with downstream induction of miR-34a and subsequent inhibition of EMP1.

Keywords: CAM assay; EMP1; TFF3; chemotherapy; miR34a; p53; retinoblastoma; tumor formation.

Figure 1

TFF3 overexpression enhances p53 transcriptional activity and protein expression and increases miR-34a expression with downstream reduction of EMP1 in Y79 RB cells. (A) Quantitative Real-time PCR confirmation of TFF3 lentiviral overexpression (Trefoil factor family peptide 3 (TFF3)) in Y79 cells compared to control cells (ctr). (B) Luciferase assays were performed with Y79 cells transiently transfected with TFF3 or empty vector control (ctr) in addition to wild-type PG13-Luc (wt PG13) or mutant MG15-Luc (mut MG15). Forced TFF3 expression leads to an increased luciferase signal upon p53 promotor activation in Y79 cells. (C) Western blot analysis showing increased p53 and TFF3 protein levels after TFF3 overexpression (TFF3). The indicated intensity ratios of p53 and TFF3 protein levels relative to β-actin levels were calculated using ImageJ software. (D,E) Quantitative real-time PCR analysis of miR-34a and EMP1 expression levels in Y79 cells compared to control cells after lentiviral TFF3 overexpression (ctr). Values are means of at least 3 independent experiments ± SEM. * p-value < 0.05, ** p-value < 0.01; statistical differences compared to the control group calculated by Student’s t-test or one-way ANOVA and Newman-Keuls post test.

Figure 2

Epithelial membrane protein 1 (EMP1) knockdown leads to reduced cell viability and proliferation and induces apoptosis in Y79 RB cells. (A) Western blot data confirmed decreased EMP1 protein levels after EMP1 knockdown (shEMP1) in Y79 cells. The CML cell line K562 served as an EMP1 positive control, ß-actin as a loading control. (B,C) Stably EMP1 knockdown Y79 RB cells (shEMP1) showed significantly decreased viability and proliferation levels compared to control cells (ctr) as revealed by (B) WST-1 assays and (C) BrdU stains. (D) EMP1 knockdown Y79 cells (shEMP1) displayed a significantly increased apoptosis rate compared to control cells (ctr) as revealed by DAPI stains. Values are means of 3 independent experiments ± SEM. *** p-value < 0.001 statistical differences compared to the control group calculated by Student’s t-test.

Figure 3

EMP1 overexpression leads to increased cell viability and proliferation and induces caspase-3/7 dependent apoptosis in Y79 RB cells. (A) Western blot data confirmed increased EMP1 protein levels after EMP1 overexpression (EMP1) in Y79 cells. The CML cell line K562 served as an EMP1 positive control, ß-actin as a loading control. (B,C) Stably EMP1 overexpressing Y79 RB cells (EMP1) showed significantly increased viability and proliferation levels compared to control cells (ctr) as revealed by (B) WST-1 assays and (C) BrdU stains. red: BrdU-labeled cells; blue: DAPI counterstaining (D) Growth curve analysis of EMP1 overexpressing Y79 RB cells showed a significant increase in cell growth rates. (E) EMP1 overexpressing Y79 cells (EMP1) displayed a significantly reduced apoptosis rate compared to control cells (ctr) as revealed by DAPI stains. (F) Caspase-3/7 activity was significantly reduced after EMP1 overexpression in Y79 RB cells (EMP1) compared to control cells (ctr). Values are means of at least 3 independent experiments ± SEM. ** p-value < 0.01; *** p-value < 0.001 statistical differences compared to the control group calculated by Student’s t-test.

Figure 4

Effect of stable EMP1 overexpression on RB cell colony formation and anchorage independent growth. (A) Quantification of colony formation assays (CFA) showing a significant higher capacity of EMP1 overexpressing Y79 RB cells to form colonies (EMP1) compared to control cells (ctr). (B) Quantification of anchorage independent growth capacity of EMP1 overexpressing Y79 RB cells (EMP1) compared to control cells (ctr) as revealed by soft agarose assay. All photographs are taken 3 weeks after seeding EMP1 overexpressing and control Y79 RB cells. Values are means of at least 3 independent experiments ± SEM. *** p-value < 0.001; statistical differences compared to the control group calculated by Student’s t-test.

Figure 5

Stable, lentiviral EMP1 overexpression increases tumor formation capacity of Y79 RB cells. (A) Photographs of CAM tumors in situ (left column), 3D tumor volume (middle column) and ruler measurements (in cm) of excised tumors (right column) revealing that tumors forming in the upper CAM after grafting EMP1 overexpressing Y79 RB cells were significantly larger compared to those developing from control cells (ctr). (B,C) Quantification of CAM assays by (B) tumor size, (C) tumor weight and (D) tumor volume. Values are means from at least 3 independent experiments (except for tumor volume which was measured exemplarily in one experimental setting) ± SEM. * p-value < 0.05, ** p-value < 0.01 statistical differences compared to the control group calculated by Student’s t-test.

Figure 6

MiR-34a and EMP1 overexpression leads to enhanced chemosensitivity. (A) Overexpression of miR-34a in Y79 RB cells leads to significantly increased apoptosis levels compared to control cells (ctr). Additional treatment with etoposide (Etop), cisplatin (CisP), or vincristine (Vin) significantly elevates the apoptosis levels compared to untreated miR-34a overexpressing Y79 RB cells. (B) Overexpression of EMP1 in Y79 RB cells leads to significantly decreased apoptosis levels compared to control cells (ctr). Additional treatment with etoposide (Etop) and vincristine (Vin) leads to higher apoptosis levels compared to untreated EMP1 overexpressing Y79 RB cells. Additional treatment with cisplatin (CisP) did not change the apoptosis level compared to untreated EMP1 expressing Y79 RB cells. Values are means of at least 3 independent experiments ± SEM. * p-value < 0.05, *** p-value < 0.001; statistical differences compared to the control group calculated by one-way ANOVA and Newman-Keuls post test.