Abstract P6-12-12: MiR-19a released by the triple negative inflammatory breast cancer SUM149 cells can be taken up by dendritic cells and induce increased synthesis of the proinflammatory cytokines IL-6 and TNF-a
Authors: Anfossi S , Burks JK , Giordano A , Cohen EN , Gao H , Debeb BG , Woodward W , Ueno NT , Ricardo AH , Hortobagyi GN , Valero V , Reuben JM 
Published: August 2014

Background: Inflammatory breast cancer (IBC) is a rare but highly aggressive form of breast cancer, responsible for 8%–10% of breast cancer–related deaths. To date, a unique molecular signature for IBC able to explain the dramatic differences in survival and tumor aggressiveness compared with non-IBC has not been identified. Triple-negative (TN) subtype is associated with worse clinical outcome in IBC patients. Tumor-associated dendritic cells (DCs) are the central regulators of immune responses in the tumor microenvironment, where the crosstalk with IBC cells may induce proinflammatory immune responses responsible of the development of highly aggressive tumor cells: cancer stem cells with epithelial mesenchymal transition (CSCs/EMT) phenotype. The two miR-19a target genes PTEN and SOCS1 regulate DC activation and maturation by inhibiting toll-like receptor (TLR) and CD40 signaling. As we found that the TN-IBC SUM149 cells expressed and secreted higher levels of miR-19a compared with the TN non-IBC SUM159 cells, we hypothesized that miR-19a secreted by SUM149 cells can be taken up by DCs and downregulate PTEN and SOCS1, leading to increased maturation of DC and production of proinflammatory cytokines upon TLR-mediated activation.

Methods: DCs were generated from healthy donor-derived monocytes cultured for 5 days in RPMI 10% FBS supplemented with IL-4 and GM-CSF 1000 U/ml. To track miRNA transfer to DCs, SUM149 cells were transfected with either Dy547-labeled non-targeting miRNA or miR-19a-mimic, then co-cultured with DCs in 1.0 mm pore size transwell chambers for 24 h. MiRNA uptake by DCs was assessed by confocal fluorescence microscopy and qRT-PCR. The effect of miR-19a uptake by DCs were assessed by measuring: the levels of PTEN and SOCS1 mRNA after 24 h of DC co-culture with miR-19a-transfected SUM149; the expression levels of the costimulatory-activation markers CD80, CD86, CD40, CD83, HLA-DR (by FACS); and the production of IL-6, TNF-α (by ELISA) after DC activation by TLR4/LPS (100 ng for 18 h). As IL-6 induces STAT3, a transcription factor for miR-19a, we also evaluated the effect of IL-6 on miR-19a expression in SUM149 cells.

Results: Fluorescent Dy547-labeled miRNA and miR-19a were transferred from SUM149 to DCs. The uptake of miR-19a induced the downregulation of both PTEN and SOCS1 mRNA in DCs, leading to increased expression of costimulatory-activation markers and production of IL-6 and TNF-α after DC activation by TLR4/LPS vs control. Stimulation of SUM149 cells with IL-6 upregulated miR-19a expression that was associated with an increased expression of CSC markers (CD44+CD24−Aldefluor+) and upregulation of EMT-regulating transcription factors.

Conclusion: DCs can uptake miR-19a secreted by SUM149 cells, leading to increased production of IL-6 and TNF-α upon DC activation by TLR stimulation. IL-6 upregulated miR-19a expression in SUM149 cells. Therefore, miR-19a/IL-6 self-sustaining loop may represent a novel way by which TN-IBC cells maintain a proinflammatory tumor microenvironment able to induce the development of tumor cells with CSCs/EMT phenotype responsible of the poor prognosis of patients with TN-IBC.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-12.