Functional Characterization of ZNF750 in esophageal squamous cell carcinoma

Abstract

Background: Esophageal squamous cell carcinoma (ESCC) is a lethal and aggressive malignancy that is a threat worldwide. It is of significant prevalence in regions of Asia including Iran, Korea, Japan and especially China. Epidemiological studies of ESCC reveal genetic variations as a risk-factor to ESCC incidence. Genomic profiling of ESCC primary tumors have already identified candidate genes involved in different cellular processes such as cell cycle and apoptosis. Data from our whole exome sequencing study of primary tumors and lymph node metastases also reveal mutations involved in these pathways; however, we also uncovered an interesting candidate involved in differentiation namely, ZNF750. ZNF750 has been reported to play important roles in other squamous cell carcinomas, and as a candidate tumor suppressor gene in ESCC.

Methods and Results: We exogenously upregulated ZNF750 expression by lentiviral system and used this model to investigate ZNF750 function in ESCC. By MTT 2D cell proliferation assay, we showed that in vitro cell proliferation in the ZNF750 group was hindered compared to controls. In addition, we set up 2D and 3D colony formation assays and found that the ZNF750 groups produced fewer colonies in 2D culture plates and produced significantly smaller colonies, when embedded in Matrigel. All of these findings are consistent with previous studies. We also studied the potential functional role of ZNF750 in 3D invasion/migration. Transwell permeable inserts were used to support a thick Matrigel culture with a nutrient gradient. Cells were embedded in Matrigel and moved along the gradient within the gel. After prolonged incubation (>2 weeks), location and size of the colonies were examined by confocal microscopy scanning. Cells with upregulated ZNF750 were found to exhibit greater mobility compared to the control cells. We also subjected ZNF750 and control groups to cisplatin to test for chemoresistance in ESCC. We found that ZNF750 expression increases chemoresistance, as opposed to the control cells.

Conclusion: From these results, ZNF750 expression inhibits cell proliferation, while enhancing cell mobility and drug resistance, all of which are characteristics of cells undergoing epithelial-mesenchymal transition (EMT). We, therefore, hypothesize that ZNF750 functions in ESSC beyond that of a tumor suppressor, but as a putative EMT regulator.

Acknowledgements: Research Grants Council Collaborative Research Fund grant number 106150246 and Asian Cancer Research Fund to MLL.