Dissection of an impact of VDR and RXRA on the genomic activity of 1,25 (OH)₂D₃ in A431 squamous cell carcinoma

Background: Human skin is the natural source, place of metabolism, and target for vitamin D-3. The classical active form of vitamin D-3, 1,25(OH)(2)D-3, expresses pluripotent properties and is intensively studied in cancer prevention and therapy. To define the specific role of vitamin D-3 receptor (VDR) and its co-receptor retinoid X receptor alpha (RXRA) in genomic regulation, VDR or RXRA genes were silenced in the squamous cell carcinoma cell line A431 and treated with 1,25(OH)(2)D-3 at long incubation time points 24 h/72 h. Extending the incubation time of A431 WT (wild-type) cells with 1,25(OH)(2)D-3 resulted in a two-fold increase in DEGs (differentially expressed genes) and a change in the amount of downregulated from 37% to 53%. VDR knockout led to a complete loss of 1,25(OH)(2)D-3-induced genome-wide gene regulation at 24 h time point, but after 72 h, 20 DEGs were found, of which 75% were downregulated, and most of them belonged to the gene ontology group "immune response". This may indicate the existence of an alternative, secondary response to 1,25(OH)(2)D-3. In contrast, treatment of A431 Delta RXRA cells with 1,25(OH)(2)D-3 for 24 h only partially affected DEGs, suggesting RXRA-independent regulation. Interestingly, overexpression of classic 1,25(OH)(2)D-3 targets, like CYP24A1 (family 24 of subfamily A of cytochrome P450 member 1) or CAMP (cathelicidin antimicrobial peptide) was found to be RXRA-independent. Also, immunofluorescence staining of A431 WT cells revealed partial VDR/RXRA colocalization after 24 h and 72 h 1,25(OH)(2)D-3 treatment.Comparison of transcriptome changes induced by 1,25(OH)(2)D-3 in normal keratinocytes vs. cancer cells showed high cell type specific expression pattern with only a few genes commonly regulated by 1,25(OH)(2)D-3. Activation of the genomic pathway at least partially reversed the expression of cancer-related genes, forming a basis for anti-cancer activates of 1,25(OH)(2)D-3. In summary, VDR or RXRA independent genomic activities of 1,25(OH)(2)D-3 suggest the involvement of alternative factors, opening new challenges in this field.