The Role of microRNA Genes in Breast Cancer Progression and Metastasis.

Abstract

Abstract MicroRNAs influence tumor progression and metastasis via action as metastasis promoting genes (for example mir-373 and mir-520) or metastasis suppressor genes (for example mir-335). To identify additional microRNAs capable of driving breast cancer metastasis in vivo, we employed a functional genomics screen using the '4T1' syngeneic (Balb/c) mouse model of breast cancer progression. Approximately 400 microRNA sequences were subcloned into the pMSCV_puro retroviral expression vector and the resultant microRNA expression library transduced into fluorescent non-metastatic 67NR-pMSCV_mCherry and mildy-metastatic 66cl4-pMSCV_mCherry cells and selected with puromycin. The transduced cell lines were then implanted into the mammary fat pads of 20 syngeneic Balb/c mice per cell line. Following establishment of primary tumors, the primary tumors and secondary organs (lung, liver, spine, femurs), including any overt metastatic deposits, were dissected under a fluorescent stereomicroscope and the microRNA inserts amplified from the integrated retroviral genome by PCR.In an alternative approach, we have studied two populations of MDA-MB-231 human breast cancer cells that exhibit different behavior in vitro and in vivo to elucidate processes involved in cellular invasion and metastasis. A late-passage MDA-MB-231 (231variant) population formed invasive clusters when embedded in Matrigel, whereas early-passage (P3) MDA-MB-231 (231) cells did not. Moreover, 231v cells were more motile than 231 cells and showed enhanced tumorigenicity when grown as subcutaneous xenografts in nude mice, whereas proliferation and apoptotic response were indistinguishable between the two lines in vitro. Molecular characterization of the two cell populations cultured in 3D by array-based gene expression profiling revealed elevated expression of NF kappa B target genes in 231v cells (IL1A, IL1B, IL6, PTX3, NR4A2, HMOX1, FLIP). MicroRNA array analysis of 3D cultures also revealed marked induction (approximately 8-fold) of the related NF kappa B target microRNAs, mir-146a and mir-146b (Figure 1). Further evaluation of a panel of human breast cancer cell lines revealed that mir-146a and mir-146b were dramatically elevated in metastatic human breast cancer cell lines that display both epithelial-to-mesenchymal transition and high NF kappa B activity (MDA-MB-231, Hs578T, MDA-MB-436), compared to non-metastatic lines. Experiments to elucidate the function of mir-146a and mir-146b during breast cancer progression are underway. Figure 1: TaqMan qPCR analysis of mir-146a and mir-146b expression in 231 and 231v cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6142.