Loss of GON4L impairs proliferation and spheroid growth in luminal breast cancer cells

Abstract

GON-4-like (GON4L) is a nuclear protein described as a putative transcriptional regulator with transcriptional repressive activity. Its oncogenic potential has been reported in bladder cancer through interactions with the transcription factor Yin Yang 1 (YY1), the corepressor SIN3 Transcription Regulator Family Member A (SIN3A), and Histone Deacetylase 1 (HDAC1). However, the functional role of GON4L in breast cancer remains largely unexplored. This study aimed to investigate the contribution of GON4L to breast cancer cell proliferation under both two-dimensional (2D) monolayer and three-dimensional (3D) culture conditions. Materials and Methods: In this study, GON4L expression in breast cancer was analyzed using publicly available datasets (UALCAN, TNMplot) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) in cell lines. GON4L was knocked-down in the MCF7 breast cancer cell line using the CRISPR-Cas9 gene editing system. The impact of GON4L loss on cellular proliferation was assessed through green fluorescent protein (GFP)-based competition and colony formation assays under 2D conditions, as well as spheroid formation assays in 3D culture systems. Statistical analyses were performed using Student's t-test. Results: GON4L expression was significantly elevated in breast cancer patient samples, including metastatic tissues. Compared with the non-tumorigenic breast epithelial cell line MCF-12A, GON4L expression was increased in MCF7 cells cultured under both 2D and 3D conditions. Genetic loss of GON4L resulted in a marked reduction in short- and long-term proliferative capacity in 2D culture systems and significantly impaired spheroid formation and growth in 3D cultures. These findings indicate that GON4L is required for sustained proliferation and maintenance of tumor-like architecture in breast cancer cells. Conclusion: Our results identify GON4L as a functionally important transcriptional regulator supporting proliferative capacity and structural organization in breast cancer cells, particularly under tumor-mimicking 3D conditions. Further investigation of GON4L-dependent regulatory mechanisms may provide insight into its potential relevance in breast cancer progression.