MicroRNA-222 reprogrammed cancer-associated fibroblasts enhance growth and metastasis of breast cancer

Abstract

Background Cancer-associated Þbroblasts (CAFs) are known to impact on tumour behaviour but mechanisms controlling this are poorly understood. Methods Breast normal fibroblasts (NFs) or CAFs were isolated from cancers by laser-microdissection or were cultured. Fibroblasts were transfected to manipulate miR-222 or Lamin B Receptor (LBR). Fibroblast conditioned medium was collected and used to treat epithelial BC lines MDA-MB-231 and MDA-MB-157. Migration, invasion, proliferation, or senescence was assessed using transwell, MTT or X-gal assays respectively. Results MiR-222 was up-regulated in CAFs as compared to NFs. Ectopic miR-222 expression in NFs induced CAF-like expression profiles, while miR-222 knock-down in CAFs inhibited CAF phenotypes. LBR was identified as a direct miR-222 target and was functionally relevant since LBR knock-down phenocopied miR-222 over-expression and LBR over-expression phenocopied miR-222 knock-down. MiR-222 over-expression, or LBR knock-down, was sufficient to induce NFs to show CAF characteristics of enhanced migration, invasion and senescence, and furthermore conditioned medium from these fibroblasts induced increased BC cell migration and invasion. The reverse manipulations in CAFs inhibited these behaviours in fibroblasts and inhibited paracrine influences on BC cells. Conclusion MiR-222/LBR have key roles in controlling pro-progression influences of CAFs in BC. This pathway may present therapeutic opportunities to inhibit CAF-induced cancer progression. 3 BACKGROUND Breast cancer (BC) is the leading cause of cancer death worldwide among women 1 , and nearly 2.3 million females are newly diagnosed annually. Although there are initial responses to treatment, many cancers relapse and distant metastases occur in nearly one third of woman; these are typically fatal. The biology behind BC metastases still remains undetermined. Therefore, understanding molecular determinants of metastasis is crucial for finding new therapeutic strategies. The tumour microenvironment consists of immune cells, blood vessels, endothelial cells, fibroblasts and extracellular matrix 4. This microenvironment plays key roles in disease outcome by inducing tumour cell proliferation and aggressiveness 5,6. Cancer-associated Þbroblasts (CAFs), an activated form of tissue-resident fibroblasts present within breast cancers, comprise a major component of the tumour microenvironment 7 , characterised most commonly by expression of-smooth muscle actin 8. CAFs can induce cancer progression 9 and metastasis 10 by secreting various cytokines, chemokines and growth factors (e.g. VEGF, FGF2, TGF , CXCL12, IL6 and IL8) 11,12 and by modulating the extracellular matrix (ECM) that facilitates tumour cell migration and invasion 13,14. CAFs also modulate immune cell function to create an immune suppressive environment during cancer progression 15. The process of transformation of CAFs from resident normal Þbroblasts is achieved by several growth factors 16,17,18 , however mechanisms of transformation have not yet been fully expl...

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