Profiling the Adrenergic System in Breast Cancer and the Development of Metastasis

Abstract

Epidemiological studies and preclinical models suggest that chronic stress might accelerate breast cancer (BC) growth and the development of metastasis via sympathetic neural mechanisms. Nevertheless, the role of each adrenergic pathway (α1, α2, and β) in human samples remains poorly depicted. Herein, we propose to characterize the profile of the sympathetic system (e.g., release of catecholamines, expression of catecholamine metabolic enzymes and adrenoreceptors) in BC patients, and ascertain its relevance in the development of distant metastasis. Our results demonstrated that BC patients exhibited increased plasma levels of catecholamines when compared with healthy donors, and this increase was more evident in BC patients with distant metastasis. Our analysis using the BC-TCGA database revealed that the genes coding the most expressed adrenoreceptors in breast tissues (ADRA2A, ADRA2C, and ADRB2, by order of expression) as well as the catecholamine synthesizing (PNMT) and degrading enzyme (MAO-A and MAO-B) genes were downregulated in BC tissues. Importantly, the expression of ADRA2A, ADRA2C, and ADRB2 was correlated with metastatic BC and BC subtypes, and thus the prognosis of the disease. Overall, we gathered evidence that under stressful conditions, both the α2- and β2-signaling pathways might work on a synergetic matter, thus paving the way for the development of new therapeutic approaches.

Keywords: adrenoreceptors; bone metastasis; breast cancer; stress; sympathetic nervous system.

Figure 1

Plasma levels of catecholamines in a cohort of BC patients. Systemic levels of NE (a) and EPI (b) CORT (c) in the BC patients (n = 72) versus healthy donors (n = 18). The circulating levels of NE (d), EPI (e) and CORT (f) at all stages of the disease were discriminated in relation to the healthy donors (n = 18 per stage). Additionally, the systemic levels of NE (g), EPI (h), and CORT (i) in the BC patients displaying no distant metastasis (M0; n = 51) versus the BC patients with distant metastasis (M1; n = 18) were also analyzed. BC patients with distant metastasis exhibiting bone metastasis only (56%) or multiple metastasis sites (44%), namely, in bone, liver, brain, lung, and skin are shown (j). The systemic levels of catecholamines, NE and EPI, and CORT were compared between BC patients displaying bone metastasis only versus multiple metastasis sites (k). p-value < 0.05 (*); <0.01 (**), <0.001 (***) was considered statistically significant.

Figure 2

Representative images of immunofluorescence analysis on bone biopsies retrieved from BC patients. TH-positive (a), α2a-positive (b), and β2-positive (c) staining. Tissue autofluorescence is denoted in green. Scale bar = 100 µm.

Figure 3

Gene expression profile of ADRs in BC tissues (using the BC dataset included in the TCGA database). (a) Heatmap of ADR expression in the TCGA-BC tissues versus their adjacent normal counterparts’ tissues. Charts comparing normal versus BC tissues for each ADR gene expression are illustrated in (b–j). The survival curves for ADRA2A (k), ADRA2C (l), and ADRB2 (m) are depicted. The tumor gene expression of ADRA2A (o), ADRA2C (p), and ADRB2 (q) in patients with localized and advanced BC is shown. p-value < 0.05 (*); <0.01 (**), <0.001 (***) was considered statistically significant.

Figure 4

Gene expression profile of ADRs by BC (using the BC dataset included in the TCGA database). (a) Heatmap of ADR gene expression in each BC subtype: luminal A, luminal b, HER2-enriched, and basal-like. The tumor gene expression of ADRA2A (b), ADRA2C (c), and ADRB2 (d) are represented in column graphs. The gene expression of ADRA2A (e), ADRA2C (f), and ADRB2 (g) is shown exclusively in the luminal-subtype patients. p-value < 0.05 (*); <0.01 (**), <0.001 (***) was considered statistically significant.

Figure 5

Gene expression profile of enzymes involved in catecholamine biosynthesis, degradation, and transportation in BC (using the BC dataset included in the TCGA database). (a) Diagram explaining the metabolism cascade of the catecholamines. (b) Heatmap of the expression markers of catecholamine metabolism. (c–j) Charts comparing normal versus BC tissues for each marker of catecholamine metabolism are illustrated (b–j). p-value < 0.05 (*), <0.001 (***) was considered statistically significant.

Figure 6

Adrenergic profile of human BC cell lines (dataset obtained from CCLE database). (a) Percentage of human BC cell line subtypes. (b) Heatmap depicting the gene expression of sympathetic markers for all human BC cells lines analyzed and categorized by each BC subtype. (c) Human BC cell lines subdivided in primary versus metastasis cell lines. (d) Gene expression of sympathetic markers on primary versus metastasis of human BC cell lines. p-value < 0.05 (*) was considered statistically significant.