Combination of vitamins A, D2 and D3 reduce tumor load and alter the expression of miRNAs that regulate genes involved with apoptosis, tumor suppression, and the epithelial-mesenchymal transition in HCT-116 colon cancer cells

Introduction: In previous work, we have shown the synergistic effects of combinations of vitamins A, D2, and D3 in reducing the proliferation of HCT-116 colon cancer cells. This combination also induced apoptosis and altered gene expression patterns as determined by transcriptomic profiling. Aims: The aims of this study were to investigate the effects of vitamins A and D in HCT-116 Crl:NU(NCr)-Foxn1nu mouse xenographs. Also, to determine potential mechanisms of action using miRNA-seq and correlated these data with results from RNA-seq.Methods: HCT-116 colon cancer cells were cultured and used for the xenograph study. Crl:NU(NCr)-Male Foxn1nu mice were injected subcutaneously with HCT-116-cells (1 X 106 in 100 µl 50% Matrigel and Sterile PBS), and after 7 days, were treated with vitamin A and D in feed for 21 days. The mice were imaged on day 28, sacrificed and the tumors were excised and measured. RNA was isolated from the HCT-116 cells and tumors, and RNA-seq and miRNA-seq were performed.Results: There was a concentration-dependent reduction in the HCT-116 colon cancer cell viability after treatment with vitamin A and D combinations. In Crl:NU(NCr)-Foxn1nu mice injected with HCT-116 colon tumor cells, treatment with vitamin A (25,000 IU) and vitamin D (4,000 IU) or vitamin A (35,000 IU) and vitamin D (5,000 IU) for 21 days significantly reduced tumor growth by ~38% and ~58%, respectively (p < 0.001). In the HCT-116 cells and excised tumors, treatment upregulated the expression of Bim, Bax, p53, and PTEN, and downregulated AXIN2, ID2 and DDX20 mRNAs, all well-known pro-apoptotic proteins, tumor suppressors, and molecules involved in the epithelial-mesenchymal transition. Ingenuity Pathway Analysis of miRNA-seq paired with RNA-seq showed correlations between miRNAs expression and expression of these genes. For example, miRNA-mRNA correlation pairing showed that treatment downregulated miR-30c-3p and miR-125b-3p which was associated with upregulated TP53. Upregulation of the tumor suppressor PTEN was associated with the downregulation of eleven miRNAs, including oncogenic miR17 and miR21.Conclusion: The results suggest that the combinations of vitamins A and D reduced the HCT-116 tumor burden in mice and altered the expression of miRNAs directly associated with genes in the apoptosis, tumor suppression, and epithelial-mesenchymal transition pathways. The data supports the hypothesis that vitamin A and D combinations impact multiple cancer signaling pathways and thus may be more effective in the treatment and prevention of cancer, as well as reducing cancer metastasis.   Keywords: apoptosis, cholcalciferol, colon cancer, DDX20, epithelial-mesenchymal transition, ergocalciferol, p53, RNA-seq, miRNA-seq, PTEN, synergism, transcriptome