Supplemental Figures Loss of circadian clock gene expression is associated with tumor progression in breast cancer Cristina Cadenas 1*, Leonie van de Sandt 2, Karolina Edlund 1, Miriam Lohr 2, Birte Hellwig 2, Rosemarie Marchan 1, Marcus Schmidt 3, Jörg Rahnenführer 2, Henrik Oster 4 and Jan G. Hengstler 1

Figure legends Supplemental Figure 1: Association of circadian genes with metastasis free survival time. High expression of CLOCK (217563_at), PER1 (202861_at), NPAS2 (205459_s_at) and RORC (206419_at) is significantly associated with longer metastasis free survival. 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. Supplemental Figure 2: Kaplan Meier plots showing metastasis survival rate of the different breast cancer subtypes, classified according to (Desmedt et al., 2008). The most aggressive subtype is the ER-/HER2- whereas the least aggressive subtype is the ER+/HER2-. Supplemental Figure 3: Association of circadian genes with metastasis free survival time in the different breast cancer molecular subtypes. A) In the ER+/HER2- breast cancer subtype (N=522) high expression of CRY2 (212696_at) is significantly associated with longer metastasis free survival time whereas high expression of TIMELESS (203046_s_at) is associated with shorter metastasis free survival. B) In the ER-/HER2- breast cancer subtype (N=140) both CLOCK (217563_at) and NPAS2 (205459_s_at) are significantly associated with better outcome. C) ARNTL2 (220658_s_at) is significantly associated with longer metastasis free survival in the HER2-overexpressing subgroup (N=104). All genes were dichotomized at the median. The log-rank test was used to assess statistical significance of the Kaplan-Meier plots. Supplemental Figure 4: Association of RNA levels of circadian genes with clinical parameters. A) For CLOCK (217563_at) only significant association with lack of metastastatic occurrence and higher age was found. B) PER1 (36829_at) associates with positive ER-status, negative HER2-status, lower grade, lack of metastasis, lower stage and higher age. C) NPAS2 (205459_s_at) was found to associate with negative ER-status and lack of metastasis and D) RORC (206419_at) behaved similar than PER1 except for lack of association with HER2-status. Differences for ER-status, HER2-status, pTstage, metastasis and age were tested by the Mann-Whitney test; differences between grading by the Kruscal-Wallis test. The analysis included 766 patients with node-negative breast cancer who have not been treated by chemotherapy.

Figure legends (continued) Supplemental Figure 5: Rational for studying correlations among clock genes in breast cancer. In all panels, the graphs at the left side represent the hypothetical expression levels of two circadian genes plotted against time. Day time is shown as a white bar (circadian/Zeitgeber times 0h to 12h), night time as black bar (circadian/Zeitgeber times 12h to 24h). The arrows indicate assumed time points of surgery for tumor tissue resection (usually at day time). These data points were taken to plot expression of gene 1 against expression of gene 2 (graphs at the right side) and correlation coefficients were calculated. Different scenarios are simulated. A: Two clock genes cycling in phase should result in a strong correlation between their RNA levels. B: A different phase-relationship between the two genes is expected to result in a lower correlation coefficient. C: Expression of two clock genes in anti-phase should yield a strong inverse correlation. D-E: Reduced levels of expression of either both (D), or one of the genes (E) do not necessarily result in loss of correlation as long as they are synchronized. F: Correlations may be lost due to loss of circadian rhythmicity. Supplemental Figure 6: Heatmap of correlations of core clock components. A correlation matrix of Spearman´s rank correlation coefficients among circadian genes is displayed as a heatmap. With exception of the perfect correlations in the diagonal (clock genes with themselves) and the two PER1 probe sets with each other, correlations between PER2 (205251_at) and PER3 (221045_s_at) and CRY2 (212695_at) and PER3 (221045_s_at) were found to be the highest (R= > 0.5) in the combined cohort of 766 node-negative breast cancer patients. Supplemental Figure 7: Correlations of circadian clock genes in tumors stratified according to ER-status, HER2- status, histological grade and metastasis occurrence. The pairwise correlation analysis shown in Fig. 4 for the combined cohort was performed as well in the Mainz (A and B), TRANSBIG (C and D) and Rotterdam (E and F) cohorts. 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. Supplemental Figure 8: Evaluation of the prognostic value of lack of coordinated expression of clock genes. The residual values in the regression plot of PER2-PER3 correlations were calculated and the association of absolute residual with prognosis was evaluated in the univariate and multivariate Cox analysis. The absolute residual showed a trend to a higher hazard ratio but did not amount to statistical significance.

Metastasis free survival rate Metastasis free survival (years) Metastasis free survival rate Metastasis free survival (years) Metastasis free survival rate Metastasis free survival (years) CLOCK > median CLOCK < median PER1 > median PER1 < median NPAS2 > median NPAS2 < median RORC > median RORC < median Metastasis free survival (years) P= 0.013P= P= P< Supplemental Fig. 1

Metastasis free survival rate Metastasis free survival (years) Supplemental Fig. 2

Metastasis free survival rate Metastasis free survival (years) Metastasis free survival rate Metastasis free survival (years) Metastasis free survival rate Metastasis free survival (years) Metastasis free survival rate Metastasis free survival (years) Metastasis free survival rate Metastasis free survival (years) Supplemental Fig. 3 A B C CRY2 > median CRY2 < median N=522 P<0.001 CLOCK > median CLOCK < median N= 140 P = TIMELESS > median TIMELESS < median N=522 P<0.001 NPAS2 > median NPAS2 < median N=140 P=0.013 ARNTL2 > median ARNTL2 < median N= 104 P = 0.01 D E

Supplemental Fig. 4 A

Supplemental Fig. 4 (continued) B

C

D

A B C D E F Supplemental Fig. 5

Supplemental Fig. 6

ER+ vs. ER- HER2- vs. HER2+ ER+ vs. ER- Grade 1+2 vs. Grade 3 No metastasis vs. early metastasis Grade 1+2 vs. Grade 3 R=0.592 R= R=0.612 R=0.228 R=0.557 R=0.216 R=0.618 R=0.286 R=0.527 R=0.324 R=0.563 R=0.314 R=0.554 R=0.278 R=0.605 R=0.304 Supplemental Fig. 7 A B

ER+ vs. ER- HER2- vs. HER2+ Grade 1+2 vs. Grade 3 ER+ vs. ER- Grade 1+2 vs. Grade 3 No metastasis vs. early metastasis R=0.544 R=0.042 R=0.554 R= R=0.535 R=0.143 R=0.487 R=0.261 R=0.488 R=0.198 R=0.534 R=0.110 R=0.489 R=0.283 R=0.530 R=0.283 Supplemental Fig. 7 (continued) C D

ER+ vs. ER- HER2- vs. HER2+ ER+ vs. ER- No metastasis vs. early metastasis R=0.499 R=0.115 R=0.533 R=0.641 R=0.571 R=0.354 R=0.349 R=0.086 R=0.430 R=0.315 R=0.461 R=0.372 E F Supplemental Fig. 7 (continued)

R=0.658 R=0.216 Grade 1 vs. Grade 3 (Mainz) PER2 (205251_at) expression PER3 (221045_at) expression variableHRP value PER PER residual abs. residual variableHRP value PER PER variableHRP value PER PER abs. residual Univariate Cox model Multivariate Cox model with PER2 and PER3 Multivariate Cox model with PER2, PER3 and absolute residual Supplemental Fig. 8