P53, miR-34a and EMP1-Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells.

Maike Busch,Stefan Klein,Harald Stephan,Nicole Dünker,Jan Große-Kreul,Oliver Scheiner,Klaus Metz

doi:10.3390/ijms20174129

Maike Busch, Stefan Klein + Show 5 more

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https://doi.org/10.3390/ijms20174129

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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.

Highlights

The retinal tumor retinoblastoma (RB) is the most common primary intraocular tumor in childhood with an incidence of 1:16,000 livebirth [1]
We performed a lentiviral overexpression of Trefoil factor family peptide 3 (TFF3) in Y79 RB cells (Figure 1A) and analyzed different potential target genes involved in TFF3 signaling to investigate the underlying mechanisms of TFF3’s tumor-suppressive functions
The function of TFF3 is controversially discussed as it is considered as a potential oncogenic factor or a tumor suppressor gene depending on the tissue investigated [26,28]

Summary

Introduction

The retinal tumor retinoblastoma (RB) is the most common primary intraocular tumor in childhood with an incidence of 1:16,000 livebirth [1]. Retinoblastoma arises in the developing retina as a part of the central nervous system (CNS) [2]. The molecular mechanisms underlying RB development have been intensively studied during the last decades and it has been postulated to be a multi-step process of progression from normal retinal tissue towards RB cells [3]. Retinal cells homozygous for RB1 loss can form benign tumors called retinomas, but further genomic changes are needed to form retinoblastoma [4]. Untreated RB will grow and extend beyond the eye and undergo metastatic spread, commonly into regional lymph nodes, bone and CNS [2]

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