Functional characterization of ZNF750 and KMT2D in esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies in the world. Sequencing studies have sought to uncover the genetic and genomic changes that occur in ESCC development. Our whole-exome sequencing study had uncovered significantly mutated genes in the primary tumor and metastatic lymph nodes, including ZNF750 and KMT2D. These genes were found to harbor silencing mutations; hence, implying possible tumor suppressive functions. The present study aimed to elucidate the relevance of ZNF750 and KMT2D in ESCC by functional characterization through multiple assays and identification of potential pathways that contribute to their functions. 3 ZNF750 is a gene reported to be mutated in squamous cell carcinomas. Mutations found were in the functional domain of this gene implying gene silencing that may disrupt its function in regulation of epithelial homeostasis leading to cancer development. In the current study, in vitro ZNF750-overexpression inhibited cell viability and clonogenicity; invasion/migration assays suggested that ZNF750 had no effect in enhancement of invasion/migration in both 2D and 3D cultures. In vivo, ZNF750-overexpression did not appear to have an effect on tumor size, but further tests are required to confirm the significance of this finding. Upon investigation of genes upregulated by ZNF750-overexpression, ZNF750 relevance in regulating epithelial homeostasis was confirmed in 2D culture, through the upregulation of epithelial and terminal differentiation genes. In addition, ZNF750-overexpression led to increased expression of p21, possibly through the p63/NOTCH/HES1 pathway. It is then hypothesized that ZNF750-induced re-expression of p21 initiates differentiation by inducing G1/S arrest, while the upregulated epithelial differentiation genes ensure the commitment of the cell to differentiation, which halts cell proliferation and eventually leads to inhibited cell growth. KMT2D is one of the most frequently mutated histone modifiers in cancers and has been implicated as exhibiting both oncogenic and tumor suppressive functions in a number of malignancies. In the present study, the CRISPR-Cas9 system and DNA transfection enabled functional characterization of the gene in ESCC cell lines. KMT2D-upregulation did not affect subcutaneous tumor size in the in vivo study; in vitro, KMT2D-upregulation did not affect 2D cell viability, clonogenicity and 3D invasion/migration. However, upon silencing of Kmt2d expression on mouse normal esophageal organoids by CRISPR-Cas9, a potential tumor suppressive function of the gene was uncovered. Kmt2d-KO organoids were found to be bigger and morphologically more irregular than the wild-type Kmt2d organoids, implying a transformational change, suggestive of a possible KMT2D function as a putative tumor suppressor gene. Taken together, the current study provides functional characterization of ZNF750 and KMT2D. The results show that ZNF750 displays tumor-suppressive capabilities through regulation of epithelial differentiation genes and the cell cycle regulator p21. Moreover, a tumor-suppressive role of KMT2D was found through induction of morphological changes in normal mouse esophageal organoids resembling transformation to malignancy in Kmt2d-KO organoids. These findings provide valuable insights into the function of these two commonly mutated genes in ESCC and further our understanding into the relevance of significantly mutated genes in ESCC development.