Tumor vascularization is a fundamental step in solid tumor progression and is orchestrated by different pathways of vasculogenesis. In malignant tumors, neoplastic cells can differentiate into endothelial-like cells acquiring the expression of endothelial markers (i.e. CD31 and CD34) and participating in the formation of vascular-like structures that functionally deliver oxygen and nutrients to the tumor site. We recently identified PDGFRβ as an important player of this process in triple negative breast cancer (TNBC). Interestingly, increasing evidence supported a connection between PDGFRβ and epithelial to mesenchymal transition (EMT), important step for the endothelial trans-differentiation process. PDGFR signaling arose as a promising target for TNBC; thus a better understanding of this new role of PDGFRβ could be relevant in the biology and treatment of TNBC. We then aimed at investigating microRNAs able to regulate tumor vascularization via PDGFRβ-mediated endothelial differentiation in TNBC, focusing on miR-9 and miR-200 family in light of their known relation with PDGFRβ and EMT.
Tube formation assay showed that in MDA-MB-231 and MDA-MB-157 TNBC cell lines transfection of miR-9 and miR-200 family members (miR-200b and miR-200c) promoted or inhibited, respectively, the ability of cells to form vascular-like structures when seeded on matrigel. Stimulation of PDGFRβ with PDGF-BB ligand induced miR-9 expression and enhanced the loop formation ability of treated cells. This advantage was, however, almost completely abrogated by the concomitant inhibition of miR-9, thus demonstrating that miR-9 contributed to PDGFRβ-mediated vasculogenic properties of TNBC. Moreover, we found that silencing of STARD13, direct target of miR-9 validated by luciferase reporter assay, improved the vasculogenic potential. Ectopic expression of miR-200 members, instead, suppressed PDGFRβ at protein level in both TNBC cell lines. Furthermore, the silencing of ZEB1, known target of miR-200 family, induced downregulation of PDGFRβ at protein and mRNA level; interestingly, the mRNA levels of ZEB1 and PDGFRβ strongly correlated in the TNBC subset of the TGCA dataset.
The in vivo effect of miR-9 and miR-200 family on vasculogenic properties of TNBC was then validated first through the generation of MDA-MB-231 clones for stable inhibition of miR-9 and restoration of miR-200c; the resulting miR-9 sponge and miR-200c xenografted tumors showed lower number of vascular lacunae than controls, identified through immunohistochemical (IHC) staining for CD31. These results were validated in orthotopic MDA-MB-231 breast tumors treated with miR-9 inhibitors or miR-200c mimics by peritumoral injection.
We finally evaluated PDGFRβ, by IHC, and miR-9 and miR-200c, by Real-time PCR, in a cohort of TNBC. IHC analysis demonstrated that PDGFRβ staining identified tumor cells participating in vascular lacunae, data further confirmed by immunofluorescence co-localization of PDGFRβ and CD31. Interestingly, unlike miR-9, the expression of miR-200c negatively associated with both the presence of vascular lacunae and tumor nests positive for PDGFRβ. Finally, in situ hybridization analysis revealed that miR-200c was mainly expressed by tumor cells.
In conclusion, our results suggest that miR-9 and miR-200 family play an opposite role in the regulation of the vasculogenic aptitude of TNBC. MiR-9 is induced by PDGFRβ and enhances the vasculogenic properties of tumor cells in part through the negative regulation of STARD13. MiR-200, instead, inhibits this aggressive phenotype indirectly suppressing PDGFRβ through targeting of ZEB1.
Citation Format: Elvira D'Ippolito, Ilaria Plantamura, Lucia Bongiovanni, Patrizia Casalini, Sara Baroni, Claudia Piovan, Rosaria Orlandi, Ambra V. Gualeni, Annunziata Gloghini, Anna Rossini, Filippo De Braud, Elda Tagliabue, Claudio Tripodo, Marilena V. Iorio. miR-9 and miR-200 regulate PDGFRβ-mediated endothelial differentiation of neoplastic cells in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A18.