Abstract (a) Background and purpose. Two major challenges in diagnosis and therapy of breast cancer lie in its heterogeneity and drug resistance. RNA helicase p68 (DDX5) has been shown to be involved in all aspects of RNA metabolism and serves as a transcriptional co-regulator. Strikingly, a recent study demonstrated a new role of p53 in modulating miRNA processing, probably through the p53-Drosha/p68 interactions. As a result, the gene expression regulated by p53-Drosha/p68 is under intensive investigation. Previous work has shown that p68 is one of the potential 6-gene predictors of breast cancer resistance to Lapatinib, a dual ErbB2/EGFR tyrosine kinase inhibitor. Furthermore, we and others have shown that p68 is involved in a complex protein-protein interacting network (e.g., Ca2+/CaM binding). This study is designed to mechanistically define the role of p68 in human breast cancer using an integrative biology approach. (b) Experimental procedures. We utilized a multifaceted strategy including cellular and molecular biology and various emerging technologies. Specifically, we measured the expression pattern of p68 at both mRNA and protein levels for a panel of ∼50 breast cancer cell lines. We performed immunohistochemistry of p68 using human breast tumor microarrays containing a total of 225 cases of normal and malignant (various grades and stages) tissues of the breast. We knocked down p68 in two representative breast cancer cell lines (SKBR3 and MDA-MB-231) and investigated their proliferation and responses to Lapatinib before and after p68 knockdown. Finally, we carried out SILAC-based proteomic profiling of these breast cancer cells after p68 knockdown and identified p68-targeted proteins and networks contributing to the drug responses. (c) Results. P68 expression pattern is distinct among different subtypes of breast cancers. More aggressive basal A and B subtypes have predominantly high p68 protein expression while low p68 expression is predominantly associated with the luminal subtype. Knockdown of p68 inhibits the proliferation of breast cancer cells and sensitize them to cancer drugs such as Lapatinib. Importantly, we identified cofilin-1, a major actin severing and -depolymerization protein, as a key target of p68 through proteomics studies. Knockdown of p68 increases cofilin expression. Cofilin has been previously implicated in platinum-resistance of ovarian cancer cells, and migration and invasion of breast cancer cells. Therefore, our results suggest that high p68 expression may contribute to drug resistance and metastasis of breast cancer cells through cytoskeletal reorganization by promoting actin polymerization. (d) Conclusions. RNA helicase p68 may contribute to personalized medicine by serving as a new molecular marker to characterize breast cancer subtypes, as a predictor for drug resistance, and as a target for a combinational therapy to circumvent drug resistance of aggressive tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2016.