Lim-Only Protein 4 (LMO4) is over-expressed in ~50% of human breast cancers in which it is associated with poor prognosis. LMO4 acts as an oncoprotein and is an important regulator of normal mammary gland development. LMO4 appears to function as a negative regulator of mammary epithelial differentiation and a positive regulator of proliferation. The molecular regulation of LMO4 and the mechanism by which LMO4 is over-expressed and contributes to oncogenesis is still unknown. Despite LMO4 being an oncogene, it is not a primary target for mutation, gene re-arrangement or amplification. Instead, over-expression of LMO4 is thought to arise from increased promoter activity due to aberrant activation of a second promoter. Thus we hypothesize that its increased expression in breast cancer may be due to aberrant transcriptional regulation.
To better understand the regulation of LMO4 we have completed a high throughput genome-wide RNA interference screen. To monitor changes in LMO4 expression, a luciferase reporter gene was placed under the control of a 10 kb regulatory region that contains two promoter regions, and human breast cancer cell line derivatives that stably expressed the LMO4 reporter were generated. The screening strategy combined a luminescent readout with a fluorescent readout to control for cell number. Candidates were filtered through three rounds of screening to identify high confidence candidates and validated in two breast cancer cell lines. The identification of regulators of LMO4 expression is critical for understanding key signalling pathways that contribute to the proliferation of breast cancer cells. Candidates that have been identified have the potential to be new therapeutic targets for breast tumours that overexpress LMO4.