NSD3-Induced Methylation of H3K36 Activates NOTCH Signaling to Drive Breast Tumor Initiation and Metastatic Progression

Histone methyltransferase NSD3 is frequently dysregulated in human cancers, yet the epigenetic role of NSD3 during cancer development remains elusive. Here we report that NSD3-induced methylation of H3K36 is crucial for breast tumor initiation and metastasis. In patients with breast cancer, elevated expression of NSD3 was associated with recurrence, distant metastasis, and poor survival. <i>In vivo</i>, NSD3 promoted malignant transformation of mammary epithelial cells, a function comparable to that of HRAS. Furthermore, NSD3 expanded breast cancer-initiating cells and promoted epithelial-mesenchymal transition to trigger tumor invasion and metastasis. Mechanistically, the long isoform (full-length transcript) of NSD3, but not its shorter isoform lacking a catalytic domain, cooperated with EZH2 and RNA polymerase II to stimulate H3K36me2/3-dependent transactivation of genes associated with NOTCH receptor cleavage, leading to nuclear accumulation of NICD and NICD-mediated transcriptional repression of E-cadherin. Furthermore, mice harboring primary and metastatic breast tumors with overexpressed NSD3 showed sensitivity to NOTCH inhibition. Together, our findings uncover the critical epigenetic role of NSD3 in the modulation of NOTCH-dependent breast tumor progression, providing a rationale for targeting the NSD3-NOTCH signaling regulatory axis in aggressive breast cancer. SIGNIFICANCE: This study demonstrates the functional significance of histone methyltransferase NSD3 in epigenetic regulation of breast cancer stemness, EMT, and metastasis, suggesting NSD3 as an actionable therapeutic target in metastatic breast cancer.