NFATc3 and PML synergistically regulate tumor-associated gene expression in a SUMOylation-Independent manner

The nuclear factor of activated T cells 3 (NFATc3) plays a significant role in various cancer-related processes, but its interactions with transcriptional modulators, particularly Promyelocytic Leukemia protein (PML), remain poorly understood. PML, a nuclear scaffold protein, is involved in tumor suppression and transcriptional regulation. This study investigates the interaction between NFATc3 and PML, focusing on the role of SUMOylation and its impact on downstream target genes. In vitro experiments, including mass spectrometry and Co-immunoprecipitation (Co-IP), were conducted to explore this interaction. Additionally, constructs with lysine-to-arginine (K→R) mutations at key SUMOylation sites were generated to determine whether PML SUMOylation is necessary for its interaction with NFATc3. We also assessed the impact of NFATc3 SUMOylation on its binding to PML. Chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qRT-PCR) were employed to measure the expression of downstream genes (Lgr5 and Olfm4) under NFATc3 and PML overexpression or knockdown conditions. Pharmacological treatment with arsenic sulfide (As₄S₄) was used to further investigate modulation of the PML-NFATc3 axis. Our findings revealed that the NFATc3-PML interaction is independent of the SUMOylation status of PML. Additionally, mutations in NFATc3 SUMOylation sites did not affect its binding to PML. The PML-NFATc3 axis regulates Lgr5 and Olfm4 expression, and co-expression of NFATc3 and PML synergistically upregulated these genes. Arsenic sulfide treatment reduced this synergistic effect, indicating its potential as a modulator. This study provides new insights into the regulatory mechanisms of NFATc3 and PML, suggesting potential therapeutic targets in cancer.