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

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.

Reduction of the tumorigenic potential of human retinoblastoma cell lines by TFF1 overexpression involves p53/caspase signaling and miR-18a regulation.

Trefoil factor family (TFF) peptides have been shown to play a pivotal role in oncogenic transformation, tumorigenesis and metastasis by changing cell proliferation, apoptosis, migration and invasion behavior of various cancer cell lines. In the study presented, we investigated the effect of TFF1 overexpression on cell growth, viability, migration and tumorigenicity of different retinoblastoma (RB) cell lines. Transient TFF1 overexpression significantly increases RB cell apoptosis levels. Stable, lentiviral TFF1 overexpression likewise decreases RB cell viability, proliferation and growth and significantly increases apoptosis as revealed by WST-1 assays, BrdU and DAPI cell counts. TFF1-induced apoptosis is executed via cleaved caspase-3 activation as revealed by caspase blockage experiments and caspase-3 immunocytochemistry. Results from pG13-luciferase reporter assays and Western blot analyses indicate that TFF1-induced apoptosis is mediated through transcriptional activity of p53 with concurrently downregulated miR-18a expression. In ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF1 overexpression significantly decreases the size of tumors forming from Y79 and RB355 cells and reduces the migration potential of RB355 cells. Differentially expressed genes and pathways involved in cancer progression were identified after TFF1 overexpression in Y79 cells by gene expression array analysis, underlining the effects on reduced tumorigenicity. TFF1 knockdown in RBL30 cells revealed caspase-3/7-independent apoptosis induction, but no changes on cell proliferation level. In summary, the in vitro and in vivo data demonstrate for the first time a tumor suppressor function of TFF1 in RB cells which is at least partly mediated by p53 activation and miR-18a downregulation.

Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines.

Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma.