Vitamin D3 Mediated Regulation of Hormone Receptors in the Pathogenesis of Triple- Negative Breast Cancer

Abstract

Introduction Breast cancer (BC) is the most ‘prevalent malignancy among women globally. Triple-negative breast cancer (TNBC), characterized by the absence of ER, PR, and HER2 expression, remains a particularly aggressive subtype with limited treatment options and poor prognosis.’ ‘Recent evidence suggests that the vitamin D receptor (VDR) and estrogen receptor beta 1 (ERβ1) may serve as tumor suppressors in TNBC.’ ‘This study investigates the therapeutic potential of modulating VDR and ERβ1 pathways using calcitriol and 17β-estradiol, respectively.’ Methods This study was conducted in three phases. ‘In Phase I, immunohistochemical analysis of VDR and ERβ1 expression was performed on 30 formalin-fixed, paraffin- embedded invasive ductal carcinoma samples, spanning 4 molecular BC subtypes.’ Phase II involved ‘molecular docking simulations to evaluate the binding affinities of calcitriol and 17β-estradiol to VDR, ERβ, EGFR, VEGF, and caspase-3 using Cresset Flare software.’ In Phase III, ‘in vitro assays using MDA-MB-468 TNBC cells were conducted to assess the effects of individual and combined treatments on cell viability (MTT assay) and expression of ERβ1, VDR, EGFR, VEGF, and caspase-3 (Immunoblotting).Results The results ‘of this study were obtained across three integrated experimental phases: immunohistochemistry, molecular docking, and in vitro functional assays, each contributing to a comprehensive understanding of the therapeutic relevance of VDR and ERβ1 in TNBC.’ Immunohistochemical (IHC) ‘findings from Phase I revealed that VDR and ERβ1 are variably expressed across molecular subtypes of BC. Notably, in TNBC cases, VDR was localized to both the cytoplasm and nucleus, whereas ERβ1 showed cytoplasmic expression only. This pattern was distinct from other subtypes such as Luminal A and B, which showed relatively higher nuclear staining, particularly for ERβ1. The exclusive cytoplasmic localization in TNBC suggests altered receptor signaling, possibly indicative of non-genomic pathways or receptor dysfunctionThese VDR and ERβ1 expression may play subtype-specific roles in tumor suppression and provide a basis for evaluating these receptors as therapeutic targets.’ In Phase II, ‘molecular docking simulations provided computational insights into the binding interactions between ligands (calcitriol and 17β-estradiol) and their target proteins. Calcitriol showed high binding affinity for VDR, confirming a strong ligand- receptor interaction at the predicted active site. Additionally, it exhibited moderate binding with EGFR and caspase-3, suggesting possible indirect regulatory effects on proliferative and apoptotic signaling. Similarly, 17β-estradiol demonstrated strong affinity for ERβ and VEGF, implicating its potential role in modulating estrogen- responsive and angiogenic pathways. These in silico findings support the therapeutic plausibility of targeting multiple signaling axes through ligand-mediated receptor activation.’ Phase III involved functional ‘validation through in vitro assays using MDA- MB-468 TNBC cells. Treatment with calcitriol (1–5 μM), 17β-estradiol (100–500 nM), and their combination significantly reduced cell viability in a dose- and time- dependent manner. Notably, the combination treatment produced greater reduction in viability compared to either agent alone, indicating potential additive or combination effects.’ Immunoblot ‘analysis further validated the molecular impact of treatment. Calcitriol exposure led to decreased ERβ1 expression and downregulation of EGFR and VEGF over time, while increasing caspase-3 levels, suggesting an induction of apoptosis. Treatment with 17β-estradiol similarly modulated ERβ1 expression, with limited effect on EGFR but a notable increase in VEGF modulation and caspase-3 downregulation. Most importantly, combined treatment resulted in the most robust molecular changes, showing simultaneous downregulation of proliferative and angiogenic markers and strong upregulation of caspase-3.’ Together, ‘these results demonstrate that dual modulation of VDR and ERβ1 using calcitriol and 17β-estradiol disrupts oncogenic signaling, promotes apoptosis, and may serve as an effective therapeutic strategy for ERβ1-positive TNBC.ABSTRACT Vitamin D3 Mediated Regulation of Hormone Receptors in the Pathogenesis of Triple- Negative Breast Cancer 3 Conclusion This study ‘demonstrates that dual targeting of VDR and ERβ1 using calcitriol and 17β-estradiol elicits favorable antitumor responses in TNBC cells. The combination strategy regulates key oncogenic pathways involved in proliferation, angiogenesis, and apoptosis, highlighting the therapeutic promise of receptor-based induced in ERβ1-positive TNBC. These findings warrant further validation in animal models and could contribute to the development of novel combination treatments for this aggressive BC subtype.’