AbstractThe hormonally active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D3), has shown to induce anti-cancerous effects through its cognate vitamin D receptor (VDR), in part through inducing the cell-cell adhesion molecule E-cadherin and inhibiting Wnt/β-catenin signalling. Melanoma is the most aggressive type of skin cancer and clinical data have shown a correlation between a decrease of VDR with shorter overall survival. Similar alterations within the VDR signalling pathway are known to occur in the context of colon cancer, in which a diminished response of 1,25D3 signal is associated with increased expression of the epithelial mesenchymal transcription factor (EMT) SNAIL1, which can directly inhibit the activities of VDR and E-cadherin, resulting in epithelial cells to lose their polarity. Additionally, approximately 66% of melanomas harbour the BRAFV600E mutation, which represents a constitutive signal of the mitogen-activated protein kinase (MAPK) that is known to phosphorylate a range of substrates, resulting in the regulation involved in hyper-proliferation and survival of transformed cells. Thus far, no reports have shown that MAPK can influence VDR function however, it is known that MAPK can phosphorylate a subgroup of nuclear receptors, which VDR is a member of, and thereby alter their signalling.
In this thesis we specifically investigated whether 1) EMT drivers, such as SNAI1, have any impact on VDR signalling and modulation of E-cadherin expression within metastatic melanoma and whether 2) the presence of a constitutively active MAPK function (BRAFV600E) have relevance to VDR signalling in metastatic melanoma.
In vitro, we show that 1,25D3 to modestly increase the expression of E-cadherin mRNA and protein in SK-MEL-28, but remained absent in A375, a cell line that expresses relatively higher levels of EMT transcription factors including SNAI1 and ZEB1. A knockdown of SNAI1 and, independently of ZEB1, in A375 cells is insufficient to recover E-cadherin expression. We also determined the absence of E-cadherin expression in A375 to not be regulated by methylation. Although A375 and SK-MEL-28 express appreciable levels of what appears to be a transcriptionally competent VDR, downstream transcriptional effects of VDR within our study remained overall muted with the exception of the 1,25D3-sensitive target gene CYP24 that catabolises 1,25D3 to its inactive form. Upon investigation on the regulation of VDR expression and function in the context of BRAFV600E vs. BRAFWT melanoma, we show for the first time that VDR mRNA and protein expression, independently, acquire dependency on BRAFV600E signalling. An abrogation of MAPK activity by the clinically applied BRAFV600E inhibitor vemurafenib (PLX4032) or MEK1/2 inhibitor PD98059 has shown to downregulate the overall VDR levels in BRAFV600E melanoma, in part by shortening the half-life of this protein involving proteasomal activity. We identified two potential MAPK acceptor sites on VDR protein, which are T175 and S177 on VDR isoform 1 and T225 and S227 on VDR isoform 2, both of which lie within the PEST region that is commonly associated with short-lived regulatory proteins mediated through the ubiquitinproteasome system. Through immunoprecipitation we confirm that VDR becomes a substrate for ubiquitin when BRAFV600E/MAPK activity is inhibited and subsequently degraded by proteasomes.
This study revealed novel findings, demonstrating that oncogenic V600E mutation elicits an acquired dependency of VDR expression, instigating an impaired VDR function in BRAFV600E melanoma. We speculate that BRAFV600E has altered VDR gene targeting by inducing VDR translocation into the cytosol and/or its association capacity with its target molecules that usually results in the abrogation of cell proliferation, including β-catenin for its cytosolic re-distribution and ubiquitin ligase FBXW7 with which VDR corporately induce the degradation of genes involves in cell cycle arrest like c-myc. We also postulate whether FBXW7, similar to the impaired regulation of VDR expression by BRAFV600E, has become altered in which the ubiquitin ligase now targets VDR for proteasomal degradation when MAPK activity is inhibited. In all of these proposed events, VDR activity associated with the anti-cancer effect becomes limited, which may explain the lack of responsiveness observed. Our study highlights the complex interactions of 1,25D3/VDR and along with the findings on the current vitamin D literature in the area, provide evidence that adequate 1,25D3 and VDR status in metastatic melanoma is not sufficient to use as an independent biomarker, but also the BRAF status and its subsequent subcellular localization and/or protein association of VDR needs be taken into consideration to determine whether vitamin D can be used as a co-treatment that hinders tumorigenic growth in melanoma.
|Date of Award||Jun 2020|
|Sponsors||David Cross Research Fund|
|Supervisor||Paul Thompson (Supervisor) & Tara Moore (Supervisor)|
- UV Radiation