Abstract
Abstract Despite advances in breast cancer treatment and prevention, metastasis remains the primary cause of breast cancer mortality. In addition to the acquisition of cellular processes in tumors that promote growth and invasion, the differentiation status of a tumor strongly predicts its metastatic potential. Differentiated tumors are generally less advanced and have better prognoses, whereas poorly differentiated tumors are aggressive and associated with a poor clinical outcome. Studies suggest that activation of stem cell-like factors in tumor cells induce an epithelial-mesenchymal transition (EMT) that promotes tumor initiating characteristics and blocks pathways promoting differentiation. Defining pathways regulating differentiation will be essential for developing therapeutic and chemopreventative strategies to inhibit breast cancer growth and metastasis. Singleminded-2s (SIM2s) is a member of the bHLH/PAS family of transcription factors that has been shown to be a regulator of differentiation. SIM2s is expressed in mammary epithelial cells and is down-regulated in human breast cancer samples. Loss of Sim2s causes aberrant mouse mammary ductal development suggestive of malignant transformation and EMT, including increased proliferation, polarity changes, and invasion into the surrounding stroma. Over-expression of Sim2s promotes differentiation in mouse mammary glands and mammary epithelial cells. These findings suggest that Sim2s is required for establishing and enhancing differentiation during normal mammary epithelial cell development and in breast cancer progression. Given that over-expression of Sim2s in the mouse mammary gland promotes epithelial differentiation, we assessed the ability of SIM2s to promote tumor cell differentiation. We stably over-expressed and depleted SIM2s in the human breast cancer cell line MCF10DCIS. COM, which is known for its diverse molecular subtypes and stem cell properties, and is used to mimic human comedo ductal carcinoma in situ. SIM2s expression levels are high in untransformed MCF10A cells, moderately expressed in MCF7 cancer cells and decreased in the more aggressive MCF10DCIS.COM line, supporting a role for SIM2s in tumor progression. Forced expression of SIM2s in MCF10DCIS.COM cells significantly inhibited growth and invasion. To evaluate whether SIM2s over-expression promoted differentiation in these cells, we performed QPCR for luminal and basal markers. SIM2s induced the expression of a broad range of luminal differentiation markers in the MCF10DCIS.COM cell line including CSN2 (b-casein), CDH2 (E-cadherin), and KER18 (keratin-18). Among the SIM2s suppressed targets, genes associated with stem cell maintenance and basal markers were found including SMO (smoothened), IHH (indian hedgehog), p63, SLUG (snail-2), KRT14 (keratin-14) and VIM (vimentin). In vivo studies showed that lack of Sim2s expression in MCF10DCIS.COM xenografts resulted in a higher occurrence of metastasis, determined by tissue staining and QPCR. We are also examining stem cell markers and characteristics through mammospheres along with flow cytometry. These new data support a role for SIM2s in normal mammary gland epithelial development and in promoting human breast tumor differentiation. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-06.
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