Differential MicroRNA Landscape Triggered by Estrogens in Cancer Associated Fibroblasts (CAFs) of Primary and Metastatic Breast Tumors

Abstract

Cancer associated fibroblasts (CAFs) play a main role in breast cancer progression and metastasis. Estrogens modulate in breast CAFs the expression of microRNAs (miRNAs) that are involved in the development of many tumors. In order to provide novel insights on the regulation of miRNAs by estrogens in breast cancer, we analyzed the expression of 754 miRNAs in CAFs obtained from primary mammary tumors and CAFs derived from a cutaneous breast cancer metastasis. Using the TaqMan™ Human MicroRNA Array, we found that 17β-estradiol (E2) modulates numerous peculiar and common miRNAs in CAFs derived from primary and the metastatic malignancies. In particular, we assessed that E2 modulates 133 miRNAs (41 up and 92 downregulated) in CAFs derived from primary breast tumors, whereas E2 modulates 415 miRNAs (399 up and 16 downregulated) in CAFs derived from a cutaneous metastasis of breast carcinoma. Therefore, a number of miRNAs three times higher in metastatic CAFs with respect to primary breast CAFs was found modulated by E2. Our findings shed new light on the cumulative regulation of miRNAs by E2 in the main players of the tumor microenvironment as CAFs. Moreover, our data may be taken into consideration that is useful toward innovative prognostic and therapeutic approaches in breast cancer progression.

Keywords: CAFs; breast cancer; estrogens; metastasis; microRNAs.

Figure 1

Volcano plot of miRNAs in cancer associated fibroblasts (CAFs) derived from primary breast tumors (A) and metastatic breast cancer (B) upon treatment with 100 nM E2 (17β-estradiol) for 4 h. The x-axis represents log2 Fold Change of miRNAs expression in estrogen stimulated samples versus vehicle treated samples, the y-axis represents –log10 p-values. The vertical dashed lines represent Fold Change = 2.0, while the horizontal dashed line represents p-value = 0.05. Points to the left (green) and right (red) of the plots represent miRNAs significantly downregulated and upregulated by E2 treatment, respectively.

Figure 2

E2-modulated the expression of miRNAs in CAFs derived from primary breast tumors. Heat Map representation of 133 E2-regulated miRNAs in CAFs treated with 100 nM E2 for 4 h and analyzed by TaqMan Low-Density Array Human miRNA. The rows represent miRNA and columns represent the treatment exposure. Each column is illustrated according to a colour scale from green (low expression) to red (high expression). The distance measured is Euclidean Distance and the clustering method is complete linkage. Dendrograms of clustering analysis for miRNAs and samples are displayed on the top and right, respectively.

Figure 3

E2-modulated expression of miRNAs in CAFs derived from cutaneous metastatic breast tumor. Heat Map representation of 138 E2-regulated miRNAs in CAFs derived from a cutaneous metastasis treated with 100 nM E2 for 4 h and analyzed by TaqMan Low-Density Array Human miRNA. Rows represent an miRNA and columns represent the treatment exposure. Each column is illustrated according to a colour scale from green (low expression) to red (high expression). The distance measured is Euclidean Distance and the clustering method is complete linkage. Dendrograms of clustering analysis for miRNAs and samples are displayed on the top and right, respectively.

Figure 4

E2-modulated expression of miRNAs in CAFs derived from cutaneous metastatic breast tumor. Heat Map representation of 138 E2-regulated miRNAs in CAFs derived from a cutaneous metastasis treated with 100 nM E2 for 4 h and analyzed by TaqMan Low-Density Array Human miRNA. Rows represent an miRNA and columns represent the treatment exposure. Each column is illustrated according to a colour scale from green (low expression) to red (high expression). The distance measured is Euclidean Distance and the clustering method is complete linkage. Dendrograms of clustering analysis for miRNA and samples are displayed on the top and right, respectively.

Figure 5

E2-modulated expression of miRNAs in CAFs derived from a cutaneous metastatic breast tumor. Heat Map representation of 139 E2-regulated miRNAs in CAFs derived from a cutaneous metastasis treated with 100 nM E2 for 4 h and analyzed by TaqMan Low-Density Array Human miRNA. Rows represent an miRNA and columns represent the treatment exposure. Each column is illustrated according to a colour scale from green (low expression) to red (high expression). The distance measured is Euclidean Distance and the clustering method is complete linkage. Dendrograms of clustering analysis for miRNAs and samples are displayed on the top and right, respectively.

Figure 6

Identification of up (n = 41) and down (n = 92) regulated miRNAs in CAFs derived from primary breast tumors and treated for 4 h with 100 nM E2.

Figure 7

Identification of up (n = 399) and down (n = 16) regulated miRNAs in CAFs derived from a cutaneous metastasis of breast tumor and treated for 4 h with 100 nM E2.

Figure 8

(A) Venn diagram of unique and joint E2-modulated miRNAs in CAFs derived from primary breast tumors and CAFs derived from a cutaneous metastasis of breast tumor. (B) up and downregulation of joint miRNAs (77) in CAFs derived from primary breast tumors and CAFs derived from a cutaneous metastasis of breast cancer and treated for 4 h with 100 nM E2.

Figure 9

Up and downregulation of unique miRNAs in CAFs derived from primary breast tumors and treated for 4 h with 100 nM E2.

Figure 10

Up and downregulation of unique miRNAs in CAFs derived from a cutaneous metastasis of breast tumor and treated for 4 h with 100 nM E2.

Figure 11

Biological function of miRNAs regulated by estrogens in CAFs derived from primary breast tumors (blue), metastatic breast tumor (black) or in both cell types (red). The analysis was performed by miRò v.2 on validated and predicted targets (number of tools predicting the interaction was at least 6).