Hierarchical paracrine interaction of breast cancer associated fibroblasts with cancer cells via hMAPK-microRNAs to drive ER-negative breast cancer phenotype

Figures & data

Figure 1. hMAPK miRNA signature associates with stromal, immune and estimate score. (A) TCGA breast cancer gene expression data set comparing hMAPK-miRNA signature to stromal, immune and estimate score. (B) ER+ breast cancer gene expression TCGA dataset comparing hMAPK-miRNA signature to stromal, immune and estimate score.

Figure 2. CAF23_BAS conditioned media leads to estrogen receptor repression. (A) Table showing the 3 different types of cancer associated fibroblasts (CAFs) cell cultures isolated from 3 different primary breast tumors. (B) Western blot data showing ER (4h) and pERK (20min) expression when MCF-7/lt E2- cells are treated with different CAF-CM. GAPDH used as loading control. (C) qPCR data showing ER mRNA expression when treated with different CAF-CM.

Figure 3. hMAPK miRNA 221/222 are involved in CAF mediated ER repression. (A) Table showing list of hMAPK miRNAs in all the CAF cell cultures vs dissociated tumor cells (DTs). (B) Treatment of MCF-7/lt E2- cells transfected with dual luciferase reporter vector with different CAF conditioned media. Data represents relative luciferase units (RLU). (C) A volcano plot showing miRNAs that are differentially expressed when MCF-7/lt E2- cells are treated with CAF23_BAS vs HMF conditioned media.

Figure 4. CAF-CM with exosomes containing hMAPK miR221/222 are involved in ER repression.(A) Western blot data showing effect of exosome depleted CAF conditioned media on pERK and ER of MCF/ltE2- cells treated with different CAF conditioned media. GAPDH used as loading control. (B) Cancer associated fibroblast-derived exosomes uptake by MCF-7/lt E2- breast cancer cells. (A) HMF exosomes. (B) CAF19 exosomes. (C) CAF21 exosomes. (D). CAF23 exosomes. (C). A volcano plot showing miRNAs that are differentially expressed in CAF23_BAS vs. CAF19_LA conditioned media.

Figure 5. Knockdown of hMAPK miR221/222 prevents CAF mediated ER repression. (A) qPCR data showing levels of miR 221 and miR 222 in CAF23_BAS cells after knockdown. (B) qPCR data showing levels of miR 221 and miR 222 in CAF23_BAS conditioned media after knockdown. (C). Western blot data showing effect on pERK and ER of MCF-7lt/E2- cells after treatment with CAF23_BAS miR221/222 knockdown conditioned media. GAPDH used as loading control d. qPCR data showing the miR221 and 222 expression in MCF-7/ltE2- cells after treatment with CAF23_BAS miR221/222 knockdown conditioned media.

Figure 6. Summary. Role of CAF in MAPK induced ER repression.