Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of 1,25D3 on estrogen receptor mediated signaling in relation to selective VDR variants.(A) The cells were treated with 100 nM of 1,25D3 for 48 h and the expressions of VDR and ERα proteins were determined. (B) The same cells were incubated with (E2) in the presence or absence of 100 nM 1,25D3 for 4 days keeping appropriate controls and subjected to the crystal violet assay. (C) The indicated cells were treated with E2 (10nM) in the presence or absence of 1 µM tamoxifen for 4 days and subjected to the crystal violet assay. The data represent analyses of two independent clones with triplicate analyses of each clone. Bars, mean ±SD; **P<0.01, ***P<0.001 (one-way ANOVA test).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3025916&req=5

pone-0016024-g004: Effects of 1,25D3 on estrogen receptor mediated signaling in relation to selective VDR variants.(A) The cells were treated with 100 nM of 1,25D3 for 48 h and the expressions of VDR and ERα proteins were determined. (B) The same cells were incubated with (E2) in the presence or absence of 100 nM 1,25D3 for 4 days keeping appropriate controls and subjected to the crystal violet assay. (C) The indicated cells were treated with E2 (10nM) in the presence or absence of 1 µM tamoxifen for 4 days and subjected to the crystal violet assay. The data represent analyses of two independent clones with triplicate analyses of each clone. Bars, mean ±SD; **P<0.01, ***P<0.001 (one-way ANOVA test).

Mentions: It is well documented that ER positive breast cancer growth is dependent on estrogen and that 1,25D3 down-regulates ERα expression in MCF-7 cells [20]. To uncover the role of VDRff and VDRFF on ERα signaling, the cells were exposed to 1,25D3 for forty eight hours and ERα protein expression was assessed. As demonstrated in Figure 4A ERα protein expression was substantially downregulated by 62% in VDRFF cells compared to 20% in Vector and VDRff cells after 1,25D3 treatment. The protein band intensities were calculated after actin normalization utilizing the UnScan-It gel program (Silk Scientific, Inc.). ERα expression was consistently downregulated in response to 1,25D3 in parental MCF-7 cells overexpressing increasing concentrations of VDRFF plasmid in contrast to increasing concentrations of VDRff plasmid at equal VDR levels (data not shown), indicating that VDRFF is more effective in mediating vitamin D action. To further identify the effects of VDR FokI polymorphism on estrogen mediated signaling, the cells were treated with estradiol in the presence or absence of 1,25D3. As shown in Figure 4B, estrogen induced cell growth was significantly inhibited by 1,25D3 in VDRFF overexpressing cells (P<0. 01) while no significant inhibition was observed in Vector and VDRff cells 4 days after treatments. Similar results were obtained 7 days after treatments (data not shown). Cumulatively, these results provide support for defining the VDRff and VDRFF variants as differential mediators of vitamin D action with the VDRFF form as the more active modulator. Next, the effect of the anti-estrogen tamoxifen was evaluated on estrogen stimulated cell proliferation as it is well known to negatively arbitrate this pathway. It is well established that tamoxifen inhibits estrogen mediated signaling by binding to ERα and thereby preventing the activation of estrogen responsive genes. Thus, Vector, VDRff and VDRFF cells were treated with estradiol in the presence or absence of tamoxifen for 4 days. As shown in Figure 4C, tamoxifen equally inhibited estrogen induced cell growth in MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF cell lines (P<0.001), whereas tamoxifen as expected, had no effect in the absence of estradiol. These results indicate that cells with the FokI polymorphism are differentially responsive only to vitamin D and not anti-estrogens.


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

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

Effects of 1,25D3 on estrogen receptor mediated signaling in relation to selective VDR variants.(A) The cells were treated with 100 nM of 1,25D3 for 48 h and the expressions of VDR and ERα proteins were determined. (B) The same cells were incubated with (E2) in the presence or absence of 100 nM 1,25D3 for 4 days keeping appropriate controls and subjected to the crystal violet assay. (C) The indicated cells were treated with E2 (10nM) in the presence or absence of 1 µM tamoxifen for 4 days and subjected to the crystal violet assay. The data represent analyses of two independent clones with triplicate analyses of each clone. Bars, mean ±SD; **P<0.01, ***P<0.001 (one-way ANOVA test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3025916&req=5

pone-0016024-g004: Effects of 1,25D3 on estrogen receptor mediated signaling in relation to selective VDR variants.(A) The cells were treated with 100 nM of 1,25D3 for 48 h and the expressions of VDR and ERα proteins were determined. (B) The same cells were incubated with (E2) in the presence or absence of 100 nM 1,25D3 for 4 days keeping appropriate controls and subjected to the crystal violet assay. (C) The indicated cells were treated with E2 (10nM) in the presence or absence of 1 µM tamoxifen for 4 days and subjected to the crystal violet assay. The data represent analyses of two independent clones with triplicate analyses of each clone. Bars, mean ±SD; **P<0.01, ***P<0.001 (one-way ANOVA test).
Mentions: It is well documented that ER positive breast cancer growth is dependent on estrogen and that 1,25D3 down-regulates ERα expression in MCF-7 cells [20]. To uncover the role of VDRff and VDRFF on ERα signaling, the cells were exposed to 1,25D3 for forty eight hours and ERα protein expression was assessed. As demonstrated in Figure 4A ERα protein expression was substantially downregulated by 62% in VDRFF cells compared to 20% in Vector and VDRff cells after 1,25D3 treatment. The protein band intensities were calculated after actin normalization utilizing the UnScan-It gel program (Silk Scientific, Inc.). ERα expression was consistently downregulated in response to 1,25D3 in parental MCF-7 cells overexpressing increasing concentrations of VDRFF plasmid in contrast to increasing concentrations of VDRff plasmid at equal VDR levels (data not shown), indicating that VDRFF is more effective in mediating vitamin D action. To further identify the effects of VDR FokI polymorphism on estrogen mediated signaling, the cells were treated with estradiol in the presence or absence of 1,25D3. As shown in Figure 4B, estrogen induced cell growth was significantly inhibited by 1,25D3 in VDRFF overexpressing cells (P<0. 01) while no significant inhibition was observed in Vector and VDRff cells 4 days after treatments. Similar results were obtained 7 days after treatments (data not shown). Cumulatively, these results provide support for defining the VDRff and VDRFF variants as differential mediators of vitamin D action with the VDRFF form as the more active modulator. Next, the effect of the anti-estrogen tamoxifen was evaluated on estrogen stimulated cell proliferation as it is well known to negatively arbitrate this pathway. It is well established that tamoxifen inhibits estrogen mediated signaling by binding to ERα and thereby preventing the activation of estrogen responsive genes. Thus, Vector, VDRff and VDRFF cells were treated with estradiol in the presence or absence of tamoxifen for 4 days. As shown in Figure 4C, tamoxifen equally inhibited estrogen induced cell growth in MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF cell lines (P<0.001), whereas tamoxifen as expected, had no effect in the absence of estradiol. These results indicate that cells with the FokI polymorphism are differentially responsive only to vitamin D and not anti-estrogens.

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