Gene Signatures of 1,25-Dihydroxyvitamin D3 Exposure in Normal and Transformed Mammary Cells

Abstract

To elucidate potential mediators of vitamin D receptor (VDR) action in breast cancer, we profiled the genomic effects of its ligand 1,25-dihydroxyvitamin D3 (1,25D) in cells derived from normal mammary tissue and breast cancer. In non-transformed hTERT-HME cells, 483 1,25D responsive entities in 42 pathways were identified, whereas in MCF7 breast cancer cells, 249 1,25D responsive entities in 31 pathways were identified. Only 21 annotated genes were commonly altered by 1,25D in both MCF7 and hTERT-HME cells. Gene set enrichment analysis highlighted eight pathways (including senescence/autophagy, TGFβ signaling, endochondral ossification, and adipogenesis) commonly altered by 1,25D in hTERT-HME and MCF7 cells. Regulation of a subset of immune (CD14, IL1RL1, MALL, CAMP, SEMA6D, TREM1, CSF1, IL33, TLR4) and metabolic (ITGB3, SLC1A1, G6PD, GLUL, HIF1A, KDR, BIRC3) genes by 1,25D was confirmed in hTERT-HME cells and similar changes were observed in another comparable non-transformed mammary cell line (HME cells). The effects of 1,25D on these genes were retained in HME cells expressing SV40 large T antigen but were selectively abrogated in HME cells expressing SV40 + RAS and in MCF7 cells. Integration of the datasets from hTERT-HME and MCF7 cells with publically available RNA-SEQ data from 1,25D treated SKBR3 breast cancer cells enabled identification of an 11-gene signature representative of 1,25D exposure in all three breast-derived cell lines. Four of these 11 genes (CYP24A1, CLMN, EFTUD1, and SERPINB1) were also identified as 1,25D responsive in human breast tumor explants, suggesting that this gene signature may prove useful as a biomarker of vitamin D exposure in breast tissue.

Keywords: BREAST CANCER; GENOMIC PROFILING; GROWTH INHIBITION; MAMMARY EPITHELIAL CELLS; MICROARRAY; VITAMIN D RECEPTOR.

Fig. 1.

Quantitative PCR analysis of selected immune gene expression in 1,25D treated hTERT-HME cells. hTERT-HME cells were treated with 100 nM 1,25D or ethanol vehicle (Con) for 24 h. RNA was isolated and real-time quantitative PCR was conducted for CD14, IL1RL1, MALL, TREM1, CAMP, SEMA6D, CSF1, IL33, TLR4, and ILlRN.The data were normalized against 18S and expressed as relative gene expression with values for control cells which were set to 1. Each bar represents mean ± standard deviation of at least three independent biological repeats analyzed in duplicate. ^*P-value <0.05 as assessed by one-tailed, unpaired t-test with Welch’s correction.

Fig. 2.

Quantitative PCR analysis of genes involved in cellular metabolism and hypoxia in 1,25D treated hTERT-HME cells. hTERT-HME cells were treated with 100 nM 1,25D or ethanol vehicle (Con) for 24 h. RNA was isolated and real-time quantitative PCR was conducted for SLC1A1,ITGB3, G6PD,PGD, HIF1A, IDH2, KDR, BIRC3,and GLUL. The data were normalized against 18S and expressed relative to values for control cells which were set to 1. Each bar represents mean ± standard deviation of at least three independent samples analyzed in duplicate. ^*P-value <0.05 as assessed by one-tailed, unpaired t-test.

Fig. 3.

Quantitative PCR analysis of VDR and CYP24A1 expression in the HME model of mammary cell transformation. HME, HME-LT, and HME-PR cells were treated with 100 nM 1,25D or ethanol vehicle (Con) for 24 h. RNA was isolated and real-time quantitative PCR was conducted for VDR and CYP24A1. The data were normalized against 18S and expressed relative to values for the vehicle treated HME parental cells. Each bar represents mean ± standard deviation of 3–4 independent samples analyzed in duplicate. ^*Bars annotated with different letters are significantly different (P-value <0.05) as assessed by one-way ANOVA. Two-way ANOVA indicated a significant interaction between cell line and treatment for both VDR and CYP24A1.

Fig. 4.

Effect of 1,25D on expression of CD14, IL1RL1, BMP6, and IL33 in HME cells and transformed derivatives. HME, HME-LT and HME-PR cells were treated with 100 nM 1,25D or ethanol vehicle (Con) for 24 h. RNA was isolated and real-time quantitative PCR was conducted for CD14, IL1RL1, BMP6, and IL33. The data were normalized against 18S and 1,25D treated values were expressed relative to vehicle treated values within each cell line. Each bar represents mean ± standard deviation of at least three independent samples analyzed in duplicate. ^*P-value <0.05 for the effect of 1,25D within each cell line as assessed by unpaired t-test. Two-way ANOVA indicated a significant interaction between cell line and treatment for the IL1RL1 and BMP6 genes.

Fig. 5.

Effect of 1,25D on expression of genes related to cellular metabolism and hypoxia in HME cells and transformed derivatives. HME, HME-LT and HME-PR cells were treated with 100 nM 1,25D or ethanol vehicle (Con) for 24 h. RNA was isolated and real-time quantitative PCR was conducted for ITGB3, SLC1A1, HIFA, BIRC3, KDR and GLUL The data were normalized against 18S and 1,25D treated values were expressed relative to vehicle treated values within each cell line. Each bar represents mean ± standard deviation of at least three independent samples analyzed in duplicate. ^*P-value <0.05 for the effect of 1,25D within each cell line as assessed by unpaired t-test. Two-way ANOVA indicated a significant interaction between cell line and treatment for the ITGB3 and SLC1A1 genes.

Fig. 6.

Effect of 1,25D on expression of CYP24A1, CD14, ILlRLl, BMP6, ITGB3, KDR, GLUL, SLClAl, and BIRC3 in MCF7 cells. MCF7 cells were treated with 100 nM 1,25D or ethanol vehicle (Con) for 24 h. RNA was isolated and real-time quantitative PCR was conducted for CYP24A1, CD14, IHRL1, BMP6, ITGB3, KDR, GLUL, SLC1A1, and BIRC3. The data were normalized against 18S and expressed relative to control values which were set to 1. Each bar represents mean ± standard deviation of three independent samples analyzed in duplicate. ^*P-value <0.05 1,25D vs. control as assessed by one-tailed, unpaired t-test.

Fig. 7.

Comparative effects of 1,25D in MCF7 and hTERT-HME cells on up-regulated genes identified as potential VDR targets by microarray analysis of 1,25D treated MCF7 cells. MCF7 and hTERT-HME cells were treated with 100 nM 1,25D or ethanol vehicle (Con) for 24 h. RNA was isolated and real-time quantitative PCR was conducted for KLK6, TRPV6, CP, and MERTK. The data were normalized against 18S and expressed relative to values for control treated MCF7 cells which were set to 1. Each bar represents mean ± standard deviation of three independent samples analyzed in duplicate. ^*Bars annotated with different letters are significantly different (P-value <0.05) as assessed by one-way ANOVA. Two-way ANOVA indicated a significant interaction between cell line and treatment for all genes.

Fig. 8.

Comparative effects of 1,25D in MCF7 and hTERT-HME cells on down-regulated genes identified as potential VDR targets by microarray analysis of 1,25D treated MCF7 cells. MCF7 and hTERT-HME cells were treated with 100 nM 1,25D or ethanol vehicle (Con) for 24 h. RNA was isolated and real-time quantitative PCR was conducted for PMP22, lL1RT, CLDN1, and CTGF. The data were normalized against 18S and expressed relative to values for control treated MCF7 cells which were set to 1. Each bar represents mean ± standard deviation of three independent samples analyzed in duplicate. ^*Bars annotated with different letters are significantly different (P-value <0.05) as assessed by one-way ANOVA. Two-way ANOVA indicated a significant interaction between cell line and treatment for the CLDN1 and CTGF genes.

Fig. 9.

Venn diagram of 1,25D regulated entities in hTERT-HME and MCF7 cells. hTERT-HME and MCF7 cells were treated for 24h with 100 nM 1,25D and processed for microarray screening on Gene ST 1.0 arrays. Data were analyzed on Gene Spring v 12.6 software and a Venn diagram was constructed for entities significantly (P< 0.05) altered with fold change of >1.5 (483 entities for hTERT-HME cells and 249 entities for MCF7 cells). The 26 entities that were similarly altered by 1,25D in both cell lines corresponded to 21 annotated genes (listed in Table IX).