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Functional significance of vitamin D receptor FokI polymorphism in human breast cancer cells.

Alimirah F, Peng X, Murillo G, Mehta RG - PLoS ONE (2011)

Bottom Line: The FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR.In response to 1α,25 (OH)(2)D(3) treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells.VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Biology, IIT Research Institute, Illinois Institute of Technology, Chicago, Illinois, United States of America.

ABSTRACT

Background: The FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids. Epidemiological studies have paradoxically implicated this polymorphism with increased breast cancer risk. 1α,25 (OH)(2)D(3), the active metabolite of vitamin D, is known to inhibit cell proliferation, induce apoptosis and potentiate differentiation in human breast cancer cells. It is well documented that 1α,25 (OH)(2)D(3) downregulates estrogen receptor α expression and inhibits estrogen mediated signaling in these cells. The functional significance of the VDR FokI polymorphism in vitamin D action is undefined.

Methods/findings: To elucidate the functional role of FokI polymorphism in breast cancer, MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF stable cell lines were established from parental MCF-7 cells as single-cell clones. In response to 1α,25 (OH)(2)D(3) treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells. The induction of the vitamin D target gene CYP24A1 mRNA was 1.8 fold higher in VDRFF cells than in VDRff cells. Estrogen receptor-α protein expression was downregulated by 62% in VDRFF cells compared to 25% in VDRff cells. VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide. PCR array analyses of VDRff and VDRFF cells revealed increased basal expression levels of pro-inflammatory genes Cyclooxygenase-2, Interleukin-8 and Chemokine (C-C Motif) Ligand 2 in MCF-7-VDRff cells by 14, 52.7 and 5 fold, respectively.

Conclusions/significance: These results suggest that a VDRff genotype may play a role in amplifying aggressive breast cancer, paving the way for understanding why some breast cancer cells respond inefficiently to vitamin D treatment.

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Comparative expression of pro-inflammatory genes in VDRff and VDRFF cells.(A) Total RNA was isolated from the cells and cDNA was subjected to qRT-PCR using primers specific to the indicated genes. The data represent analyses of three independent experiments. Bars, mean ±SD; **P<0.01, ***P<0.001 (one-way ANOVA test). (B) The cells were treated with 100 nM of 1,25D3 for 24 h and total RNA was evaluated for the expression of selected genes using qRT-PCR. The data represent analyses of three independent experiments. Bars, mean ±SD; *P<0.05, **P<0.01, ***P<0.001(one-way ANOVA test).
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pone-0016024-g006: Comparative expression of pro-inflammatory genes in VDRff and VDRFF cells.(A) Total RNA was isolated from the cells and cDNA was subjected to qRT-PCR using primers specific to the indicated genes. The data represent analyses of three independent experiments. Bars, mean ±SD; **P<0.01, ***P<0.001 (one-way ANOVA test). (B) The cells were treated with 100 nM of 1,25D3 for 24 h and total RNA was evaluated for the expression of selected genes using qRT-PCR. The data represent analyses of three independent experiments. Bars, mean ±SD; *P<0.05, **P<0.01, ***P<0.001(one-way ANOVA test).

Mentions: The results presented thus far characterized VDRff and VDRFF as distinct, diversely modulating 1,25D3 action. Therefore, a Signal Transduction Pathway Finder PCR Array (SA, Biosciences) was employed to ascertain whether these VDR alleles differentially regulate any genes in the signal transduction pathway independent of 1,25D3 treatment. Table 1 shows various differentially expressed genes with fold changes of 2.5 or higher in VDRff cells compared to their VDRFF counterpart. Notably, the pro-inflammatory genes Cyclooxygenase-2 (COX-2/PTGS2) [23], Interleukin-8 (IL-8) [24] and Chemokine C-C Motif Ligand 2 (CCL2/MCP-1) [25] were upregulated 14, 52, and 5 fold respectively in VDRff cells compared to VDRFF cells. Additionally, the apoptosis suppressor Baculoviral IAP repeat containing 3 (BIRC-3/cIAP2) [26] was upregulated 8 fold. The differential expression of these genes in FF and ff variants was confirmed by qRT-PCR. As illustrated in Figure 6A, basal upregulation of several pro-inflammatory genes in VDRff expressing cells was observed compared to VDRFF. These genes include COX-2 (P<0.001), IL-8 (P<0.001), CCL2 (P<0.001) and BIRC-3 (P<0.001). The effect of 1,25D3 on the COX-2, IL-8, CCL2 and BIRC-3 genes was also assessed in the three cell lines. As illustrated in Figure 6B, 1,25D3 treatment of VDRFF cells significantly downregulated the expression of IL-8 (P<0.001, VDRFF control vs. treatment), CCL2 (P<0.001, VDRFF control vs. treatment) and BIRC-3(P<0.001, VDRFF control vs. treatment). In VDRff cells, only CCL2 (P<0.01) and BIRC-3(P<0.05) were downregulated in response to 1,25D3 treatment. Unexpectedly, however, COX-2 mRNA was not significantly downregulated in VDRFF cells in response to 1,25D3.


Functional significance of vitamin D receptor FokI polymorphism in human breast cancer cells.

Alimirah F, Peng X, Murillo G, Mehta RG - PLoS ONE (2011)

Comparative expression of pro-inflammatory genes in VDRff and VDRFF cells.(A) Total RNA was isolated from the cells and cDNA was subjected to qRT-PCR using primers specific to the indicated genes. The data represent analyses of three independent experiments. Bars, mean ±SD; **P<0.01, ***P<0.001 (one-way ANOVA test). (B) The cells were treated with 100 nM of 1,25D3 for 24 h and total RNA was evaluated for the expression of selected genes using qRT-PCR. The data represent analyses of three independent experiments. Bars, mean ±SD; *P<0.05, **P<0.01, ***P<0.001(one-way ANOVA test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3025916&req=5

pone-0016024-g006: Comparative expression of pro-inflammatory genes in VDRff and VDRFF cells.(A) Total RNA was isolated from the cells and cDNA was subjected to qRT-PCR using primers specific to the indicated genes. The data represent analyses of three independent experiments. Bars, mean ±SD; **P<0.01, ***P<0.001 (one-way ANOVA test). (B) The cells were treated with 100 nM of 1,25D3 for 24 h and total RNA was evaluated for the expression of selected genes using qRT-PCR. The data represent analyses of three independent experiments. Bars, mean ±SD; *P<0.05, **P<0.01, ***P<0.001(one-way ANOVA test).
Mentions: The results presented thus far characterized VDRff and VDRFF as distinct, diversely modulating 1,25D3 action. Therefore, a Signal Transduction Pathway Finder PCR Array (SA, Biosciences) was employed to ascertain whether these VDR alleles differentially regulate any genes in the signal transduction pathway independent of 1,25D3 treatment. Table 1 shows various differentially expressed genes with fold changes of 2.5 or higher in VDRff cells compared to their VDRFF counterpart. Notably, the pro-inflammatory genes Cyclooxygenase-2 (COX-2/PTGS2) [23], Interleukin-8 (IL-8) [24] and Chemokine C-C Motif Ligand 2 (CCL2/MCP-1) [25] were upregulated 14, 52, and 5 fold respectively in VDRff cells compared to VDRFF cells. Additionally, the apoptosis suppressor Baculoviral IAP repeat containing 3 (BIRC-3/cIAP2) [26] was upregulated 8 fold. The differential expression of these genes in FF and ff variants was confirmed by qRT-PCR. As illustrated in Figure 6A, basal upregulation of several pro-inflammatory genes in VDRff expressing cells was observed compared to VDRFF. These genes include COX-2 (P<0.001), IL-8 (P<0.001), CCL2 (P<0.001) and BIRC-3 (P<0.001). The effect of 1,25D3 on the COX-2, IL-8, CCL2 and BIRC-3 genes was also assessed in the three cell lines. As illustrated in Figure 6B, 1,25D3 treatment of VDRFF cells significantly downregulated the expression of IL-8 (P<0.001, VDRFF control vs. treatment), CCL2 (P<0.001, VDRFF control vs. treatment) and BIRC-3(P<0.001, VDRFF control vs. treatment). In VDRff cells, only CCL2 (P<0.01) and BIRC-3(P<0.05) were downregulated in response to 1,25D3 treatment. Unexpectedly, however, COX-2 mRNA was not significantly downregulated in VDRFF cells in response to 1,25D3.

Bottom Line: The FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR.In response to 1α,25 (OH)(2)D(3) treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells.VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Biology, IIT Research Institute, Illinois Institute of Technology, Chicago, Illinois, United States of America.

ABSTRACT

Background: The FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids. Epidemiological studies have paradoxically implicated this polymorphism with increased breast cancer risk. 1α,25 (OH)(2)D(3), the active metabolite of vitamin D, is known to inhibit cell proliferation, induce apoptosis and potentiate differentiation in human breast cancer cells. It is well documented that 1α,25 (OH)(2)D(3) downregulates estrogen receptor α expression and inhibits estrogen mediated signaling in these cells. The functional significance of the VDR FokI polymorphism in vitamin D action is undefined.

Methods/findings: To elucidate the functional role of FokI polymorphism in breast cancer, MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF stable cell lines were established from parental MCF-7 cells as single-cell clones. In response to 1α,25 (OH)(2)D(3) treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells. The induction of the vitamin D target gene CYP24A1 mRNA was 1.8 fold higher in VDRFF cells than in VDRff cells. Estrogen receptor-α protein expression was downregulated by 62% in VDRFF cells compared to 25% in VDRff cells. VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide. PCR array analyses of VDRff and VDRFF cells revealed increased basal expression levels of pro-inflammatory genes Cyclooxygenase-2, Interleukin-8 and Chemokine (C-C Motif) Ligand 2 in MCF-7-VDRff cells by 14, 52.7 and 5 fold, respectively.

Conclusions/significance: These results suggest that a VDRff genotype may play a role in amplifying aggressive breast cancer, paving the way for understanding why some breast cancer cells respond inefficiently to vitamin D treatment.

Show MeSH
Related in: MedlinePlus