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, 14 (2), R41

Aromatase Inhibitors, Estrogens and Musculoskeletal Pain: Estrogen-Dependent T-cell Leukemia 1A (TCL1A) Gene-Mediated Regulation of Cytokine Expression

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Aromatase Inhibitors, Estrogens and Musculoskeletal Pain: Estrogen-Dependent T-cell Leukemia 1A (TCL1A) Gene-Mediated Regulation of Cytokine Expression

Mohan Liu et al. Breast Cancer Res.

Abstract

Introduction: Arthralgias and myalgias are major side effects associated with aromatase inhibitor (AI) therapy of breast cancer. In a recent genome-wide association study, we identified SNPs - including one that created an estrogen response element near the 3' end of the T-cell leukemia 1A (TCL1A) gene - that were associated with musculoskeletal pain in women on adjuvant AI therapy for breast cancer. We also showed estrogen-dependent, SNP-modulated variation in TCL1A expression and, in preliminary experiments, showed that TCL1A could induce IL-17RA expression. In the present study, we set out to determine whether these SNPs might influence cytokine expression and effect more widely, and, if so, to explore the mechanism of TCL1A-related AI-induced side effects.

Methods: The functional genomic experiments performed included determinations of TCL1A, cytokine and cytokine receptor expression in response to estrogen treatment of U2OS cells and lymphoblastoid cell lines that had been stably transfected with estrogen receptor alpha. Changes in mRNA and protein expression after gene knockdown and overexpression were also determined, as was NF-κB transcriptional activity.

Results: Estradiol (E2) increased TCL1A expression and, in a TCL1A SNP-dependent fashion, also altered the expression of IL-17, IL-17RA, IL-12, IL-12RB2 and IL-1R2. TCL1A expression was higher in E2-treated lymphoblastoid cell lines with variant SNP genotypes, and induction of the expression of cytokine and cytokine receptor genes was mediated by TCL1A. Finally, estrogen receptor alpha blockade with ICI-182,780 in the presence of E2 resulted in greatly increased NF-κB transcriptional activity, but only in cells that carried variant SNP genotypes. These results linked variant TCL1A SNP sequences that are associated with AI-dependent musculoskeletal pain with increased E2-dependent TCL1A expression and with downstream alterations in cytokine and cytokine receptor expression as well as NF-κB transcriptional activity.

Conclusions: SNPs near the 3' terminus of TCL1A were associated with AI-dependent musculoskeletal pain. E2 induced SNP-dependent TCL1A expression, which in turn altered IL-17, IL-17RA, IL-12, IL-12RB2, and IL-1R2 expression as well as NF-κB transcriptional activity. These results provide a pharmacogenomic explanation for a clinically important adverse drug reaction as well as insights into a novel estrogen-dependent mechanism for the modulation of cytokine and cytokine receptor expression.

Figures

Figure 1
Figure 1
Effect of estradiol concentration on mRNA expression for cytokine receptors in lymphoblastoid cell line SNPs. Effect of estradiol (E2) concentration on mRNA expression for (A) T-cell leukemia 1A (TCL1A), (B) IL-17RA, (C) IL-17, (D) IL-12RB2, (E) IL-12 and (F) IL-1R2 in Human Variation Panel lymphoblastoid cell lines with wild type (WT) (n = 3) or variant (V) (n = 3) SNP sequences. *P < 0.05 and **P < 0.001 compared with zero E2, +P < 0.05 and ++P < 0.01 for differences between WT and V SNP genotypes at the same E2 concentrations.
Figure 2
Figure 2
T-cell leukemia 1A knockdown and overexpression and IL-17RA and IL-17 expression. (A) Relative T-cell leukemia 1A (TCL1A), IL-17RA and IL-17 mRNA expression levels in U2OS-estrogen receptor alpha (ERα) cells after TCL1A was knocked down using four different specific siRNAs. (B) Western blots of TCL1A, IL-17RA, IL-17 and β-actin, with or without TCL1A knockdown in U2OS-ERα cells. (C) Relative TCL1A, IL-17RA and IL-17 mRNA expression levels after TCL1A overexpression (OE) in U2OS-ERα cells. (D) Western blots of TCL1A, IL-17RA, IL-17 and β-actin, with or without TCL1A overexpression in U2OS-ERα cells. All data are the mean ± standard error of the mean of triplicate determinations.
Figure 3
Figure 3
Estradiol upregulates T-cell leukemia 1A and IL-17RA in series. (A) T-cell leukemia 1A (TCL1A) and IL-17RA expression after TCL1A was knocked down in U2OS-estrogen receptor alpha (ERα) cells by four different TCL1A-specific siRNAs and by a pool of the four siRNAs. (B) TCL1A and IL-17RA expression after TCL1A was knocked down by four different TCL1A-specific siRNAs and a pool of the four siRNAs with an additional 24 hours exposure to 0.1 nM estradiol (E2). All values represent the mean ± standard error of the mean of triplicate determinations.
Figure 4
Figure 4
Estradiol upregulates T-cell leukemia 1A upstream of IL-12, IL-12RB2 and IL-1R2. (A) T-cell leukemia 1A (TCL1A), IL-12, IL-12RB2 and IL-1R2 expression in U2OS-estrogen receptor alpha (ERα) cells after TCL1A was knocked down (KD) by an siRNA pool, with or without subsequent exposure to 0.1 nM estradiol (E2) for 24 hours. (B) TCL1A, IL-12, IL-12RB2 and IL-1R2 expression in U2OS-ERα cells after TCL1A overexpression (OE). All values are the mean ± standard error of the mean of triplicate determinations. (C) Diagrammatic representation of the sequential E2 upregulation of TCL1A followed by altered cytokine or cytokine receptor expression.
Figure 5
Figure 5
T-cell leukemia 1A knockdown and NF-κB transcriptional activity. (A) T-cell leukemia 1A (TCL1A) expression in U2OS-estrogen receptor alpha (ERα) cells before and after siRNA knockdown (KD), with or without the addition of 0.1 nM estradiol (E2) for an additional 24 hours. (B) NF-κB transcriptional activity after co-transfection with an NF-κB reporter construct together with TCL1A-negative siRNA or TCL1A-specific siRNA. Forty-eight hours after transfection, the cells were exposed to 0.1 nM E2 for an additional 24 hours. All values are the mean ± standard error of the mean of triplicate determinations. (C) Western blots for TCL1A, phosphorylated IκB, IκB and actin after TCL1A knockdown in U2OS-ERα cells, with or without the addition of 0.1 nM E2 for an additional 24 hours.
Figure 6
Figure 6
SNP-related variation in T-cell leukemia 1A expression and NF-κB activity in lymphoblastoid cell lines. SNP-related variation in T-cell leukemia 1A (TCL1A) expression and NF-κB transcriptional activity in three lymphoblastoid cell lines (LCLs) with variant (V) genotypes and three LCLs with wild type (WT) genotypes for the chromosome-14 SNPs after exposure to increasing concentrations of estradiol (E2), or E2 plus the estrogen receptor (ER) antagonist ICI-182,780. (A) Quantitative RT-PCR of TCL1A expression after exposure to increasing concentrations of E2 for 24 hours, followed by 0.01 nM E2 plus increasing concentrations of ICI-182,780 for an additional 24 hours. (B) The same six LCLs were transfected with an NF-κB reporter construct. After exposure to increasing concentrations of E2 for 24 hours, followed by 0.01 nM E2 plus increasing concentrations of ICI-182,780 for an additional 24 hours, luciferase activities were determined. All values represent the mean ± standard error of the mean of triplicate determinations.

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