The vitamin D3 metabolite, 25-hydroxyvitamin D3, demonstrates activity in VDRhigh non-small cell lung cancer cells independent of CYP27B1
Verone-Boyle, Alissa R.
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Lung cancer is the leading cause of cancer-related deaths in the United States. Each year, more individuals die from lung cancer than breast, prostate, and colon cancers combined. The National Lung Cancer Screening Trial has recently demonstrated that low dose helical CT scans rather than chest X-rays are more successful at identifying early-stage, smaller nodules. As a result, CT scans are being implemented as a method for lung cancer screening. Despite the advances in screening, the five-year survival of lung cancer patients is a mere 17%. Thus, a need exists for identifying new agents to be used in the lung cancer setting, either alone or in combination with current therapies. Epidemiologic studies have identified a role for the serum vitamin D3 metabolite, 25-hydroxyvitamin D3 (25D3) in the lung cancer setting. Elevated serum 25D3 concentrations are associated with a lower risk of developing lung cancer. Further, serum 25D3 concentrations predict overall survival specifically in early-stage non-small cell lung cancer (NSCLC) patients. These studies and others highlight the potential clinical relevance of 25D3 in lung cancer. However, experiments to test vitamin D3 as a potential therapeutic agent in lung cancer have thus far only focused on the active metabolite, 1,25-dihydroxyvitamin D3 (1,25D3). To date, no data exist on the biological role 25D3 plays in lung cancer despite overwhelming epidemiologic evidence 25D3 is a key factor in the disease. We have previously identified a phenotype in lung cancer cells that predicts responsiveness to the active metabolite, 1,25D3. NSCLC cells expressing higher basal levels of the vitamin D receptor (VDR) and low levels of the 1,25D3 catabolizing enzyme CYP24A1 are more sensitive to 1,25D3 treatment, compared to cells with low VDR and high CYP24A1 expression. Cells expressing higher VDR (VDRhigh) also frequently harbor EGFR mutations. We hypothesized that NSCLC cells harboring EGFR mutations may represent a molecularly defined phenotype that is responsive to vitamin D3 treatment. Specifically, we hypothesized that 25D3 may exert similar anti-cancer activities as observed with 1,25D3 in VDRhigh EGFR mutant cells. VDRhigh NSCLC cell lines were found to respond to 25D3 treatment in vitro by inducing VDR target gene expression and inhibiting clonogenic growth. Induction of VDR target genes by 25D3 is dependent on the presence of VDR. In vivo , dietary vitamin D3 supplementation significantly elevated 25D3 and suppressed the growth of VDRhigh NSCLC xenografts. Mice with higher serum 25D3 concentrations (68ng/mL) displayed overall lower tumor volumes, and grew at a slower rate, compared to mice with low serum 25D3 (12ng/mL). Beneficial effects of correcting low vitamin D concentrations were also observed: tumor growth rates were statistically slower. Although elevating serum 25D3 concentrations specifically in EGFR mutant VDRhigh cells is an effective approach to inhibit lung cancer cell growth in vitro and in vivo , no studies have been conducted to test if VDR protein, the receptor for both 25D3 and 1,25D3, is even present in EGFR mutant NSCLCs. Therefore, a lung cancer tissue microarray consisting of never-smoking lung cancer patients (a population where EGFR mutations are commonly observed) was stained for VDR. 48 cases on the TMA had matching DNA available to perform targeted DNA sequencing for EGFR mutations. Cases harboring deletions in exon 19 of EGFR expressed higher VDR protein, compared to cases harboring L858R mutations. The observation that tumors harboring EGFR exon 19 deletions also express higher VDR is particularly intriguing because patients with exon 19 deletions have a better prognosis and overall response rate to EGFR targeted therapies than patients with L858R mutations. Thus, the VDR is present and targetable in EGFR mutant NSCLCs. In lung cancer, genes involved in vitamin D3 metabolism are often dysregulated. CYP27B1, the enzyme responsible for 25D3 conversion to 1,25D3, is variably expressed in lung tumors and often lost during disease progression. Therefore, determining if NSCLC cells require CYP27B1 conversion to 1,25D3 is a rate limiting factor in potentially applying 25D3 in a clinical situation. To determine if 25D3 could induce vitamin D3 signaling alone, we utilized methods to chemically (pharmacologic inhibitors) or genetically (zinc finger nucleases, siRNA) inactivate CYP27B1 and test if 25D3 still induced VDR target gene transcription. CYP27B1 activity was assessed by measuring 1,25D3 production via LC-MS/MS. Under conditions where no 1,25D3 was produced, indicating near complete CYP27B1 inactivation, 25D3 treatment continued to induce VDR target gene mRNA levels. These data indicate that 25D3 is an active VDR ligand in NSCLC cells and can signal independently of CYP27B1 expression. Thus, loss of CYP27B1 will not preclude the use of vitamin D3 supplements in NSCLC patients. Finally, the transcriptome induced by 25D3 compared to 1,25D3 were investigated by performing an RNA sequencing experiment in VDRhigh EGFR mutant cells. 25D3 was found to regulate pathways involved in progression through the cell cycle, inflammatory pathways including NFκB and cell metabolism, which we had anticipated. Novel pathways regulated by 25D3 specifically were also identified, such as regulation of the mismatch repair pathway. Additionally, both 25D3 and 1,25D3 were found to regulate multiple pathways involving translation and peptide elongation, suggesting a primary role for vitamin D3 in protein synthesis. Thus, 25D3 elicits a wide range of biological functions in VDRhigh NSCLC cells that may contribute to the potent anti-tumor responses we observed. However, we have only just begun to establish the full spectrum of how 25D3 functions in NSCLC cells. Nonetheless, the results presented here establish 25D3 as an active VDR ligand in EGFR mutant NSCLC cells, regardless of CYP27B1 activity. Furthermore, we have established a molecular phenotype for patients whom are likely to respond to 25D3 treatment via dietary vitamin D3 supplementation.