Thyronamine regulation of TAAR1 expression in breast cancer cells and investigation of its influence on viability and migration

Abstract

Objectives

A correlation exists between breast cancer and thyroid disorders, which are common in elderly women. Thyroid hormones are degraded into trace amines, which can bind to the G-protein-coupled receptor trace amine-associated receptor 1 (TAAR1) and thereby activate it. The transformation of thyroid hormones into trace amines is carried out by the ornithine decarboxylase. Previously, we showed that TAAR1 overexpression (IRS ≥6) was associated with a significantly longer OS in primary breast cancer patients during a long-term follow-up of up to 14 years. Aim of the present study was to analyze the regulation of TAAR1 in breast cancer cell lines and the influence of triiodothyronine (T₃), thyronamines, and tetraiodothyroacetic acid (Tetrac) on the expression of TAAR1 in breast cancer cells.

Methods

The effect of T₃, thyronamines, and Tetrac on the expression of TAAR1 in breast cancer cell lines MCF-7 and T47D was analyzed via PCR and Western blot. A MTT assay was performed to test the metabolic cell viability. A scratch assay was performed to analyze cell migration.

Results

Stimulation of MCF-7 cells with 10 nM 3-iodothyronamine (T₁AM) significantly increased TAAR1 protein expression (P=0.008). In T47D cells, TAAR1 expression was significantly upregulated after the addition of 10 µg/mL estradiol to 10 nM T₁AM (P=0.008). A significant (P=0.028) reduction in MCF-7 cell viability through the incubation with T₁AM could be detected. Cell migration of MCF cells was significantly reduced through incubation with 10 nM T₁AM.

Conclusion

A significant upregulation of TAAR1 induced by stimulation with T₁AM may be a sign for an increased decarboxylation of thyroid hormones in breast cancer cells. In addition, there seems to be an influence of estradiol for the T₁AM-induced upregulation of TAAR1 in T47D cells. TAAR1-related cell transduction mechanisms seem to be an interesting target for endocrine treatment options of breast cancer patients.

Keywords:

• breast cancer
• TAAR1
• 3-iodothyronamine
• Tetrac
• MCF7
• T47D

Supplementary materials

Figure S1 Real-time TaqMan^® PCR in MCF7- and T47D- cells after stimulation with T1AM.

Notes: (A) Bar chart as a relative representation of TAAR1 mRNA expression in MCF7 cells after incubation with T₁AM for 2h detected via Real-time TaqMan^® PCR. 0.01nM T₁AM induced a significant upregulation of TAAR1 expression (P=0.043). 0.1nM T₁AM showed no significant influence on TAAR1 expression (P=0.398). (B) Bar chart of TAAR1 mRNA expression in T47D cells after incubation with T1AM for 2h detected via Real-time TaqMan PCR. 0.1nM T₁AM induced a significant upregulation of TAAR1 expression (P=0.028). 0.01nM T₁AM showed no significant influence on TAAR1 expression (P=0.144). This bar graph shows the mean of relative TAAR1 expression; therefore, the presentation of error bars is not appropriate. *P<0.05.
Abbreviation: T1AM, 3-iodothyronamine.

Figure S2 Real-time TaqMan^® PCR in MCF7- and T47D- cells after stimulation with Tetrac.

Notes: (A) Bar chart of TAAR1 mRNA expression in MCF7 cells after incubation with Tetrac detected via Real-time TaqMan^® PCR. 0.1nM Tetrac for 2h led to a significant increase of TAAR1 expression in MCF7 cells (P=0.017). 0.01nM Tetrac showed no significant influence on TAAR1 expression (P=0.345). (B) Bar chart of TAAR1 mRNA expression in T47D cells after incubation with Tetrac for 2h detected via Real-time TaqMan PCR. 0.1nM Tetrac induced a significant upregulation of TAAR1 expression (P=0.043). 0.01nM Tetrac showed no significant influence on TAAR1 expression (P=0.917). This bar graph shows the mean of relative TAAR1 expression; therefore, the presentation of error bars is not appropriate. *P<0.05.
Abbreviation: Tetrac, tetraiodothyroacetic acid.

Figure S3 Western Blot analysis of TAAR1 protein expression in MCF7 and T47D cells after stimulation with T₁AM.

Notes: (A) Bar chart of TAAR1 expression in MCF7 cells after incubation with 10nM T1AM for 2h. T1AM induced a significant upregulation of TAAR1 protein expression (P=0.008). 1 nM T₁AM showed no significant influence on TAAR1 protein expression (P=0.767). (B) Bar chart of TAAR1 expression in T47D cells after incubation with 1nM and 10nM T₁AM for 2h. No significant change in TAAR1 protein expression could be observed (1nM: P=0.441; 10nM: P=0.678). This bar graph shows the mean of relative TAAR1 expression; therefore, the presentation of error bars is not appropriate.
Abbreviation: T1AM, 3-iodothyronamine.

Acknowledgments

In memory, we thank Sandra Schulze for her excellent technical assistance in this article. The study was funded by the Bayerische Gleichstellungsförderung grant (2016/2017) of Bavaria, Germany, and by the Foundation of the Frankfurter Allgemeinen Zeitung (FAZIT) foundation grant (2017/2018) of Germany.

Author contributions

AV, UJ, ET, and ND conceived and designed the experiments. ET, CK, and SiH performed the experiments. AV, UJ, ET, CK, SiH, and KH analyzed the data. ET, AV, UJ, and KH wrote the research article. SM, KH, SH, HH, DM, NH, RW, and ND interpreted the results and revised the manuscript. All authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.