Loss of circadian clock gene expression is associated with tumor progression in breast cancer

Abstract

Several studies suggest a link between circadian rhythm disturbances and tumorigenesis. However, the association between circadian clock genes and prognosis in breast cancer has not been systematically studied. Therefore, we examined the expression of 17 clock components in tumors from 766 node-negative breast cancer patients that were untreated in both neoadjuvant and adjuvant settings. In addition, their association with metastasis-free survival (MFS) and correlation to clinicopathological parameters were investigated. Aiming to estimate functionality of the clockwork, we studied clock gene expression relationships by correlation analysis. Higher expression of several clock genes (e.g., CLOCK, PER1, PER2, PER3, CRY2, NPAS2 and RORC) was found to be associated with longer MFS in univariate Cox regression analyses (HR<1 and FDR-adjusted P < 0.05). Stratification according to molecular subtype revealed prognostic relevance for PER1, PER3, CRY2 and NFIL3 in the ER+/HER2- subgroup, CLOCK and NPAS2 in the ER-/HER2- subtype, and ARNTL2 in HER2+ breast cancer. In the multivariate Cox model, only PER3 (HR = 0.66; P = 0.016) and RORC (HR = 0.42; P = 0.003) were found to be associated with survival outcome independent of established clinicopathological parameters. Pairwise correlations between functionally-related clock genes (e.g., PER2-PER3 and CRY2-PER3) were stronger in ER+, HER2- and low-grade carcinomas; whereas, weaker correlation coefficients were observed in ER- and HER2+ tumors, high-grade tumors and tumors that progressed to metastatic disease. In conclusion, loss of clock genes is associated with worse prognosis in breast cancer. Coordinated co-expression of clock genes, indicative of a functional circadian clock, is maintained in ER+, HER2-, low grade and non-metastasizing tumors but is compromised in more aggressive carcinomas.

Keywords: ARNTL/2, aryl hydrocarbon receptor nuclear translocator-like/2; BHLHE40/41, basic helix-loop-helix family, member e; CLOCK, circadian locomotor output cycles kaput; CRY1/2, cryptochrome circadian clock 1/2; DBP, D site of albumin promoter (albumin D-box) binding protein; DFS, disease-free survival; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; HR, hazard ratio; MFS, metastasis-free survival; NFIL3, nuclear factor, interleukin 3 regulated; NPAS2, neuronal PAS domain protein 2; NR1D2, nuclear receptor subfamily 1, group D, member 2; PER1/2/3, period circadian clock 1/2/3; RORA/B/C, RAR-related orphan receptor alpha/beta/gamma; SCN, suprachiasmatic nucleus; breast cancer; circadian clock; clock genes; estrogen receptor; metastasis-free survival; tumor progression.

Figure 1.

Association of PER2, PER3, CRY2 and TIMELESS with metastasis-free survival time. High expression of PER2 (205251_at), PER3 (221045_s_at) and CRY2 (212695_at) is associated with longer metastasis free survival time, whereas high TIMELESS (203046_s_at) expression is associated with shorter metastasis free survival time. The analysis included 766 patients with node-negative breast cancer who have not been treated by chemotherapy. All genes were dichotomized at the median. The log-rank test was used to assess statistical significance of the Kaplan-Meier plots. Kaplan Meier analysis of further circadian genes is shown in Fig. S1.

Figure 2.

Association of RNA levels of PER2, PER3, CRY2 and TIMELESS with clinical parameters. Significant associations (P < 0.05) of PER2 (205251_at), PER3 (221045_s_at) and CRY2 (212695_at) with positive estrogen receptor (ER) status, lower grade, lack of metastasis, lower pT stage (1) and higher age were obtained. In addition, PER3 and CRY2 also associate with negative HER2-status. TIMELESS (203046_s_at), in contrast, showed significant association with negative ER-status, positive HER2-status higher grade, higher pT stage (2+3) and early metastasis. Differences for age, ER status, HER2 status, pTstage and metastasis were tested by the Mann-Whitney test, differences between grading by the Kruskal-Wallis test. The analysis included 766 patients with node negative breast carcinoma. Further analyses are shown in Fig. S4.

Figure 3.

Correlation of PER2, PER3, CRY2 and TIMELESS with the proliferation and the estrogen receptor (ER) metagenes. PER2 (205251_at), PER3 (221045_s_at) and CRY2 (212695_at) show a negative correlation with the proliferation metagene and a positive correlation with the ER metagene. Conversely, TIMELESS (203046_s_at) shows a positive correlation with the proliferation metagene but negative correlation with the ER metagene. Scatterplots along with the corresponding Spearman´s rank correlation coefficients are shown. The analysis included carcinomas of 766 patients with node-negative breast cancer.

Figure 4.

Correlations of circadian clock genes in tumors stratified according to ER status, HER2 status, histological grade and metastasis occurrence. Correlation coefficients between PER2 (205251_at) and PER3 (221045_s_at) and between CRY2 (212695_at) and PER3 (221045_s_at) are higher in estrogen receptor (ER)-positive and HER2-status negative tumors, and also in tumors with low grade and with no metastatic occurrence (green color). Conversely, correlations are lower in ER negative, HER2 status positive, high grade and metastatic tumors (red color). Correlations were analyzed using the Spearman correlation test. The subgroup analysis included the combined cohort of 766 patients with node-negative breast cancer except in grade, since grade information is only available for the Mainz and the Transbig cohorts (combined N = 480). Correlation analysis in the single cohorts is shown in Fig. S7.