Poster S3 - 24R,25(OH)2D3 Mediates Laryngeal Cancer Cell Tumorigenesis via a Membrane-Associated Mechanism through a Complex of Receptors

Introduction: Laryngeal cancer is the most common type of head and neck cancer with more than 75% of patients diagnosed in a late stage. The larynx is a secondary sex organ that has been shown to be estrogen-responsive. Vitamin D3 supplementation is becoming widely accepted as a therapeutic in many common estrogen-responsive cancers. 24R,25-dihydroxy vitamin D3 (24,25), an active metabolite of vitamin D3, has been shown to have an anti-tumorigenic effect in some breast cancers as well as some laryngeal cancers, which was dependent on the expression of estrogen receptor alpha-66 (ERα66). 24,25 has been shown to regulate tumorigenesis via a membrane-associated receptor thought to be located within the cholesterol-enriched caveolae, the structure of which caveolin-1 (cav-1) plays a major role. Nuclear vitamin D receptor (nVDR) and PDIA3 are membrane-associated receptors for 1,25(OH)2D3 suspected to reside within the caveolae. The nuclear VDR is associated with the caveolae membrane via palmitoylation; when activated, it dimerizes with the vitamin A receptor and passes through the nuclear membrane to affect proliferation and metastasis gene transcription. Some studies demonstrated the interaction of PDIA3 and nVDR within the caveolae in response to treatment with the other active vitamin D metabolite 1,25(OH)2D3. There are reports that 24,25 can also bind the nVDR, but whether it acts through the nVDR and/or PDIA3 is not known. The goal of this study was to determine the mechanism driving the effects of 24,25 in estrogen-responsive laryngeal cancer tumorigenesis, and if it is mediated by caveolae and potential receptors within the caveolae.

Materials and

Methods: ER66+ UM-SCC-12 (UM12) and ER66- UM-SCC-11A (UM11A) cells lines were transfected with a lentivirus packaged shRNA targeting the cav-1 gene. Western blot analysis was used to validate that cav-1 had been silenced in both cell lines. Cells were cultured until confluence in 24 or 96-well plates, then pre-treated with methyl-beta cyclodextrin (MBC) for 30 minutes and then with 24,25 for 15 minutes to determine how destroying the caveolae would change the effect of 24,25 on apoptosis via p53 production and proliferation via EdU assay. After 15 minutes, the media were aspirated, and fresh full media were replaced. Silenced cav-1 cells were treated with vehicle or 24,25 for 15 minutes and assessed for proliferation and p53 protein levels 24 hours later. To investigate which receptors are involved in the mechanism driving the effect of 24,25, cells were assessed for the gene expression via qPCR and protein levels via western blot analysis of each putative receptor. The mechanism was then assessed by first treating with either an nVDR inhibitor (MeTC7) or a PDIA3 inhibitor (E64FC26) for 30 minutes and then treated with vehicle or 24,25 for 15 minutes to evaluate the role of nVDR and PDIA3 respectively in 24,25’s effect on proliferation and apoptosis at 24 hours. To evaluate the role of palmitoylation on 24,25’s effect, cells were treated with the inhibitor, 2-bromopalmitate for 30 minutes and then an added vehicle or 24,25 treatment for an additional 15 minutes. Cells were then assessed for proliferation and apoptosis at 24 hours.

Results, Conclusions, and Discussions: shCav1 UM12 cells showed a 90.3% reduction while shCav1 UM11A cells showed a 72.9% reduction in caveolin-1 protein compared to wild type cells. MBC blocked 24,25’s effect on the proliferation in UM12 and UM11A cells. MBC did not alter 24,25’s effect on apoptosis in either UM12 or UM11A cells. Interestingly, 24,25’s effects on proliferation and apoptosis in both cell types were blocked by silencing cav-1. Protein levels of the nVDR was higher in the UM11A cells compared to the UM12s. However, qPCR expression levels were similar in both cell lines. MeTC7 blocked 24,25 mediated proliferation in UM11As but not UM12s. The nVDR inhibitor, MeTC7, completely blocks 24,25’s effect on apoptosis in UM12s and UM11As. Similar PDIA3 levels were found in both UM12 and UM11A cells measured by both mRNA expression and western blot. The PDIA3 inhibitor, E64FC26 blocks 24,25’s effect on proliferation and apoptosis in UM12s and only apoptosis in UM11As. Inhibition of palmitoylation blocked the effect of 24,25 on both proliferation and apoptosis in both cell lines. The present study suggests that the caveolae and caveolin-1 play a major role in the effect of 24,25 on laryngeal cancer tumorigenesis. These data suggest that the putative 24,25 receptors nVDR and PDIA3 mediate the effects of 24,25 on proliferation and apoptosis. Inhibition of palmitoylation suggested that the effect of 24,25 involves the palmitoylation of a receptor to the membrane. In summary, 24,25 regulates laryngeal cancer via a mechanism involving a complex of receptors within the caveolae involving a palmitoylated receptor. These data suggest a major role of 24R,25-dihydroxyvitamin D3 and its membrane associated signaling pathway regulating in laryngeal cancer.

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