Dormancy signatures and metastasis in estrogen receptor positive and negative breast cancer

Abstract

Breast cancers can recur after removal of the primary tumor and treatment to eliminate remaining tumor cells. Recurrence may occur after long periods of time during which there are no clinical symptoms. Tumor cell dormancy may explain these prolonged periods of asymptomatic residual disease and treatment resistance. We generated a dormancy gene signature from published experimental models and applied it to both breast cancer cell line expression data as well as four published clinical studies of primary breast cancers. We found that estrogen receptor (ER) positive breast cell lines and primary tumors have significantly higher dormancy signature scores (P<0.0000001) than ER- cell lines and tumors. In addition, a stratified analysis combining all ER+ tumors in four studies indicated 2.1 times higher hazard of recurrence among patients whose tumors had low dormancy scores (LDS) compared to those whose tumors had high dormancy scores (HDS) (p<0.000005). The trend was shown in all four individual studies. Suppression of two dormancy genes, BHLHE41 and NR2F1, resulted in increased in vivo growth of ER positive MCF7 cells. The patient data analysis suggests that disseminated ER positive tumor cells carrying a dormancy signature are more likely to undergo prolonged dormancy before resuming metastatic growth. Furthermore, genes identified with this approach might provide insight into the mechanisms of dormancy onset and maintenance as well as dormancy models using human breast cancer cell lines.

Figure 1. Clustering of dormancy signature scores.

A) Dormancy score analysis in breast cancer cell lines. The cell lines are ordered by dormancy scores (low to high from left to right). The rows correspond to genes and the columns represent cell lines. Expression levels for positive dormancy genes (upregulated genes - top section) and negative dormancy genes (downregulated genes - bottom section) were clustered by a hierarchical clustering algorithm. The colors represent log₂ fold change compared to the average from −2.5 (blue, below average) to +2.5 (red, above average) with white as the average value. A, B, and L stand for Basal A, Basal B, and Luminal classifications, respectively. B) Correlation of cell line dormancy scores with proliferation indices from Table 1 of with ER- lines plotted as squares and ER+ lines plotted as diamonds. Straight line fits of ER- (blue, Spearman correlation coefficient r = .027) and ER+ (red, r = −0.76) cell lines are plotted. The ER status of HCC1500 is unclear (ATCC indicates it as ER+ while it is ER- by gene expression and Western blot in [8]) and it was not used in the analysis. MDA-MB-231 (large orange square), MCF7 (large green diamond) and T47D (large yellow diamond) are identified. C) Patient tumor analysis. The four clinical studies were clustered as in A. In the ER status bar, ER status is indicated by black (ER+), blue (ER−) or white (not determined) bars. The two genes for which probes were not present in the van de Vijver et al. data set are represented by gray bars. D) Comparison of clustering of cell lines and patient data. Top: Positive dormancy genes that are upregulated in high dormancy score cell lines or patients. Bottom: Negative dormancy genes that are up regulated in low dormancy score cell lines or patients.

Figure 2. Metastasis-free analysis for four clinical studies.

(A, B) van de Vijver et al., (C, D) Loi et al., (E,F) Wang et al., (G, H) Pawitan et al. Kaplan Meier estimates of metastasis-free proportion among patients with high (upper third, green), medium (middle third, red), and low (bottom third, black) dormancy scores for patients with ER+ (A,C,E,F) and ER− (B,D,F,G) tumors.

Figure 3. RNAi suppression of dormancy upregulated genes accelerates tumor take and growth of ER+ luminal MCF-7 cells.

(A) Percent of tumor take at the indicated time points after injecting with 4 10⁶ MCF7 cells treated with the indicated siRNAs in the mouse mammary fat pad. (B) Tumor volume (mm³) at 12 days for tumors generated by MCF7 cells treated with the indicated siRNAs and injected in the mouse mammary fat pad. (C–D) Q-PCR analysis for the expression of BHLHE41 (C) and NR2F1 (D) mRNAs after 48 hrs of treatment with control or specific siRNAs targeting these mRNAs.