Background <p>CK2 is a pleiotropic, heterotetrameric kinase elevated in numerous cancers and, as such, has garnered much attention as a target for novel antineoplastic agents. Currently, silmitasertib, a selective CK2 inhibitor, has an orphan designation for medulloblastoma and neuroblastoma. Tissue-specific overexpression of the catalytic subunit, CK2α, is sufficient to drive the development of mammary tumors in mice, underlying its potential role in breast oncogenesis. Our lab has demonstrated that CK2β protein expression is associated with endocrine resistance and that targeted disruption of CK2 activity results in ERα proteolysis. Among the factors driving endocrine resistance is overexpression of HER2/EGFR, which contributes to both proliferation and phenotypic plasticity in ER⁺ breast cancer. Here, we sought to determine the impact of CK2 targeting on HER2/EGFR signaling, phenotypic plasticity, and cell proliferation in endocrine-sensitive and endocrine-resistant breast cancer. Furthermore, we explored the potential of combined lapatinib and CK2-based therapies in decreasing breast cancer proliferation and epithelial-mesenchymal transition (EMT).</p> Methods <p>CK2 subunit expression and function were investigated in ERα-mutant, tamoxifen-resistant MCF-7 Y537S breast cancer cells and additional breast cancer subtypes. CK2 was inhibited pharmacologically using CX-4945 or genetically via shRNA knockdown of CK2β. Cell proliferation, spheroid growth, migration, and EMT markers were assessed using live-cell imaging, wound-healing assays, immunoblotting, and qRT-PCR. Publicly available patient datasets were analyzed to evaluate the prognostic relevance of <i>CSNK2B</i> expression.</p> Results <p>CK2β expression was significantly upregulated in ERα-mutant breast cancer cells and patient tumors. Pharmacological inhibition or genetic depletion of CK2β suppressed proliferation, spheroid formation, and migration, accompanied by reduced ERK phosphorylation and cyclin D1 expression. CK2β inhibition also attenuated EMT, marked by decreased N-cadherin, vimentin, and EMT transcription factors, while enhancing E-cadherin expression. High <i>CSNK2B</i> expression correlated with poorer relapse-free survival, particularly in ERα⁺ and HER2-E breast cancers. Notably, CK2β inhibition sensitized breast cancer cells to HER2-targeted therapy.</p> Conclusions <p>CK2 is a critical regulator of endocrine-resistant breast cancer progression, promoting proliferation, EMT, and therapeutic resistance through ERK/cyclin D1 signaling. Inhibition of CK2 potentiated the effects of lapatinib across several cell lines. Furthermore, shRNA-mediated knockdown of the CK2β subunit phenocopies pharmacological disruption of CK2 and highlights the significance of the regulatory subunit in breast cancer cell biology. Targeting CK2, and, more selectively, CK2β represent a promising approach to overcome endocrine resistance and enhance responsiveness to HER2-directed treatments.</p>