miR-9-Mediated Inhibition of EFEMP1 Contributes to the Acquisition of Pro-Tumoral Properties in Normal Fibroblasts

Abstract

Tumor growth and invasion occurs through a dynamic interaction between cancer and stromal cells, which support an aggressive niche. MicroRNAs are thought to act as tumor messengers to "corrupt" stromal cells. We previously demonstrated that miR-9, a known metastamiR, is released by triple negative breast cancer (TNBC) cells to enhance the transition of normal fibroblasts (NFs) into cancer-associated fibroblast (CAF)-like cells. EGF containing fibulin extracellular matrix protein 1 (EFEMP1), which encodes for the ECM glycoprotein fibulin-3, emerged as a miR-9 putative target upon miRNA's exogenous upmodulation in NFs. Here we explored the impact of EFEMP1 downmodulation on fibroblast's acquisition of CAF-like features, and how this phenotype influences neoplastic cells to gain chemoresistance. Indeed, upon miR-9 overexpression in NFs, EFEMP1 resulted downmodulated, both at RNA and protein levels. The luciferase reporter assay showed that miR-9 directly targets EFEMP1 and its silencing recapitulates miR-9-induced pro-tumoral phenotype in fibroblasts. In particular, EFEMP1 siRNA-transfected (si-EFEMP1) fibroblasts have an increased ability to migrate and invade. Moreover, TNBC cells conditioned with the supernatant of NFs transfected with miR-9 or si-EFEMP1 became more resistant to cisplatin. Overall, our results demonstrate that miR-9/EFEMP1 axis is crucial for the conversion of NFs to CAF-like cells under TNBC signaling.

Keywords: EFEMP1; cancer-associated fibroblasts; chemoresistance; miR-9; miRNA; triple-negative breast cancer; tumor microenvironment.

Figure 1

EFEMP1 is downregulated in breast cancer-associated and TNBC-conditioned fibroblasts. In-silico evaluation of EFEMP1 levels in paired NFs/CAFs of six breast cancer patients (a); in normal human dermal fibroblasts conditioned with the supernatants of breast cancer cells of different subtypes (b) and early passage of primary CAFs isolated from human breast cancer samples classified as ER+ (n = 7), TNBC (n = 7) and HER2+ (n = 6) (c).

Figure 2

EFEMP1 is a direct target of miR-9. Evaluation of EFEMP1 gene and protein levels by qRT-PCR (a), western blot (b) and IHC (c). qRT-PCR and western blot analysis were performed on NFs miR-9 vs. control. Protein expression levels are indicated above western blot bands. IHC images show fibulin-3 expression in ex vivo samples of tumors grown from the co-injection of MDA-MB-468 cells and NFs miR-NEG/9. Images are representative; the experiment was performed on 6 tumors per group. Scale bars 2.5 μm (d). Luciferase assay performed on HEK293 cell line transfected with miR-9 or control and with wild-type or mutated EFEMP1 3′UTR (mutated sequence shown above). Data are presented as the mean of three biological replicates ±SEM (*** p < 0.001, ns = non-significant).

Figure 3

EFEMP1 silencing increases fibroblast’s motility. Migration (a), invasion (b) and wound healing (c) assays performed on fibroblasts transfected with miR-9 (wound healing exclusively)/si-EFEMP1 or controls. In Figure 3c, the red line identifies the region of the wound which is still not occupied by cells. Images are representative and data are presented as mean of three biological replicates ±SEM. (* p < 0.05; ** p < 0.01); scale bars, 100 μm.

Figure 4

NFs miR-9/siEFEMP1 reduce tumor cell sensitivity to cisplatin. MDA-MB-468 cell count upon treatment with cisplatin (24 h) after 24h of conditioning with the supernatant of (a) NFs miR-9 or (b) si-EFEMP1, compared to controls. Cell count data are presented as mean of the percentage of viable treated (CISP = cisplatin) cells of three biological replicates, compared to non-treated (NT) cells, ±SEM (** p < 0.01, *** p < 0.001).

Figure 5

miR-9 expression in TNBC correlates with different CAF subsets and resistance to cisplatin. (a) Bubble plot showing computed Pearson correlations between TNBC subgroups according to miR-9 expression, CAF subsets and biological signatures of related functions. ns: non-significant, significant p value < 0.05. Bubble colour represents Person correlation, while size corresponds to −log10 p value, as illustrated in plot legend. (b) BRCA1 gene expression in two well-characterized cohorts of patients with TNBC treated in neoadjuvance with cisplatin (GSE18864 and GSE103668), evaluated Pathological complete response by Miller-Payne (MP) criteria (0: MP 0, 1, 2 Progression, no change or still high tumor cellularity; 1: MP 3, 4 minor and marked loss of tumor cells, 2: MP 5 non-malignant cells) (left panel), and BRCA1 gene expression in TNBC sub-grouped by miR-9 expression (right panel).