Exploiting Vitamin D Receptor and Its Ligands to Target Squamous Cell Carcinomas of the Head and Neck

**Background:** Vitamin D and its receptor (VDR) have been investigated in many cancers, but knowledge for head and neck cancer (HNC) is limited. HNC is among the top ten most common cancers worldwide and frequently exhibits limited treatment response due to chemoresistance. This study aimed to investigate the (pre)clinical and therapeutic relevance of the VDR/VitD-axis in HNC.

**Methods:** The study included a case-control analysis of 40 newly diagnosed HNC patients and 40 age-sex-matched healthy controls from Egypt. Serum 25-hydroxyvitamin D levels were quantified by HPLC. VDR expression in tumor biopsies was analyzed by immunofluorescence and immunohistochemistry. Bioinformatic analysis of the TCGA HNC cohort (n=604) was performed. In vitro experiments used multiple HNC cell lines (FaDu, HNCUM-02T, Pica, SCC-4), including a cisplatin-resistant Pica cell line (Pica~res~) established by six months of sub-toxic cisplatin exposure. VDR nuclear translocation was quantified using high-content screening microscopy. Cell viability was assessed by CellTiter-Glo assay, and drug synergy was determined using the Chou-Talalay combination index (CI) method. 3D tumor spheroids were generated using ultra-low adhesion culture. RNA sequencing and Ingenuity Pathway Analysis were used to profile nuclear receptor expression and signaling pathways.

**Key Results:** VitD serum levels were lowest in patients with poorly differentiated cancers (4.1 ± 0.5 ng/mL), increasing from moderate (7.3 ± 4.3 ng/mL) to well-differentiated (13.2 ± 3.4 ng/mL) tumors. The mean serum VitD level was 7.4 ± 4.5 ng/mL in cancer patients versus 28.7 ± 4.6 ng/mL in healthy individuals. Females showed higher VitD insufficiency compared to males, correlating with poor tumor differentiation. VDR expression was inversely correlated with VitD levels and was highest in poorly differentiated, highly proliferative (Ki67-high) tumors. TCGA analysis confirmed VDR overexpression in HNC tumor versus non-tumor tissues (n=564, p=0.0059), with significant correlation to histological differentiation (n=540, p=0.0002) and perineural invasion (n=393, p=0.0006). VDR expression was significantly increased in HPV-negative HNC patients (n=114, p<0.0001). RXRα expression did not significantly correlate with these clinical parameters. In vitro, VitD (100 nM) induced dose-dependent VDR nuclear translocation. The Chou-Talalay algorithm demonstrated synergistic killing (CI < 1) for VitD/cisplatin combinations in all tested HNC cell lines. In 3D tumor spheroids, VitD alone impaired spheroid formation, and VitD/cisplatin cotreatment significantly reduced spheroid area and viability, with more prominent effects in cisplatin-resistant cells. Mechanistically, VitD/cisplatin cotreatment decreased expression of phosphorylated mTOR and Akt, whereas cisplatin alone did not significantly reduce these activated signaling proteins.

**Clinical Implications:** This study provides evidence that VitD deficiency is severe in HNC patients (mean 7.4 ng/mL) and correlates with poor tumor differentiation. The synergistic enhancement of cisplatin efficacy by VitD, particularly through PI3K/Akt/mTOR pathway downregulation, suggests that VitD supplementation during primary (radio)chemotherapy could improve treatment outcomes. Gender-specific differences in VitD levels, potentially influenced by socioeconomic and cultural factors in the MENA region, warrant consideration in future supplementation strategies. The findings support further (pre)clinical investigation of VDR/VitD-targeted drug combinations for HNC.