MicroRNA-510 mediated negative regulation of Caveolin-1 in fibroblasts promotes aggressive tumor growth

Abstract

Introduction: In the US, despite the recent decline in breast cancer deaths, a persistent mortality disparity exists between black and white women with breast cancer, with black women having a 41% higher death rate. Several studies are now reporting that racial disparities can exist independent of socioeconomic and standard of care issues, suggesting that biological factors may be involved. Caveolin-1 (Cav1) loss in the tumor stromal compartment is a novel clinical biomarker for predicting poor outcome in breast cancer including triple negative subtype, however the mechanism of Cav1 loss is unknown. We previously identified miR-510-5p as a novel oncomir and propose here that the high levels observed in patients is a novel mechanism leading to stromal Cav1 loss and worse outcomes.

Methods: Cav1 was identified as a direct target of miR-510-5p through luciferase, western blot and qPCR assays. Stromal cross talk between epithelial cells and fibroblasts was assessed in vitro using transwell co-culture assays and in vivo using xenograft assays.

Results: We found that Cav1 is a direct target of miR-510-5p and that expression in fibroblasts results in an 'activated' phenotype. We propose that this could be important in the context of cancer disparities as we also observed increased levels of circulating miR-510-5p and reduced levels of stromal Cav1 in black women compared to white women with breast cancer. Finally, we observed a significant increase in tumor growth when tumor cells were co-injected with miR-510-5p expressing cancer associated fibroblasts in vivo.

Conclusion: We propose that miR-510-5p mediated negative regulation of Cav1 in fibroblasts is a novel mechanism of aggressive tumor growth and may be a driver of breast cancer disparity.

Keywords: Caveolin-1; breast cancer; disparities; microRNA; stroma.

Figure 1

miR-510-5p directly targets CAV1 and is racially disparate in breast cancer. (A) Caveolin-1 (CAV1) mRNA stromal levels in African American (AA) and Caucasian (CA) breast cancer patients. Adapted from Chang Data set, Oncomine. (B) Quantitation of miR-510-5p levels from serum of AA and CA benign and cancer breast patients. (C) Schematic representation of miR-510-5p binding site and complementary seed sequence (upper case letters) within the 3’UTR of human CAV1. Luciferase activity (D) and qPCR analysis (E) of HEK293 cells transiently co-transfected with CAV1 3’UTR or mutated (CAV1 SDM) and miR-510-5p or scrambled control (scr). **p<0.01; ***p<0.005.

Figure 2

miR-510-5p inhibits Cav1 protein expression in fibroblasts. Western blot (A) and qPCR (B) analysis of Caveolin-1 and (C) miR-510-5p levels in human WPMY1 cells transfected with increasing concentrations of miR-510-5p or scrambled (scr) control. Western blot (D) and qPCR (E) analysis of Caveolin-1 and (F) miR-510-5p levels in mouse 3T3 cells transfected with increasing concentrations of miR-510-5p or scrambled (scr) control. (D: lower panel) Schematic representation of miR-510-5p binding site and complementary seed sequence (upper case letters) within the 3’UTR of mouse CAV1. *p<0.05; ***p<0.005; ****p<0.001; ns, not significant.

Figure 3

miR-510-5p activates fibroblasts and promotes cell migration. qPCR (A) and western blot (B) analysis of WPMY1 cells co-cultured with MDA 231 cells stably transfected with either miR-510-5p or scrambled (scr) control. qPCR (C) and western blot (D) analysis of WPMY1 cells treated with conditioned media from MDA 231 cells stably transfected with either miR-510-5p or scrambled (scr) control. (E) qPCR analysis and (F) Transwell migration assay of WPMY1 cells co-cultured with MDA 231 cells transfected with either miR-510-5p (231/510) or scrambled control (231/scr). *p<0.05; **p<0.01.

Figure 4

miR-510-5p expressing fibroblasts promote tumor growth. qPCR analysis of (A) epithelial and fibroblast markers and (B) COL1A1 and miR-510-5p levels in CAFs isolated from orthotopic tumors. (C) Tumor growth, (D) Tumor weight and (F) Cav1 quantitation of tumors resulting from orthotopic injections of MDA 231 cells either alone (2 x 10⁵) or together (1.5 x 10⁵ 231 + 5 x 10⁴ CAFs) with scr (231:scr CAF) or 510 (231:510 CAF) CAFs into female nude mice. (E) Representative 20X magnification images of Cav1 IHC staining. (G) Quantification of Cav1 protein levels in fibroblasts from orthotopic tumors. *p<0.05; ***p<0.005; ns, not significant.