An in vivo genome-wide RNAi screen identifies novel mediators of paclitaxel response in breast cancer

Abstract

Background: Inflammatory breast cancer (IBC) is an aggressive form of locally advanced breast cancer with elevated metastatic potential, characterized by the frequent presence of tumor emboli in dermal and parenchymal lymph vessels. This study examines the role of TGFb signaling in the molecular biology of IBC. Methods: TGFb1 induced cell motility (i.e. xCELLigence, wound healing assays) and gene expression (RNA sequencing) were investigated in a panel of three IBC and three subtype-matched nIBC cell lines. In addition, a series of tissue samples from 75 and 135patients with and without IBC was investigated for nuclear expression of total SMAD2, SMAD3 and SMAD4 using immunohistochemistry. SMAD protein expression data were related to gene expression data from patients with available Affymetrix HGU133plus2 profiles. Results: TGFb1-induced cell motility was strongly abrogated in all IBC cells (P ¼ 0.003), independent of their molecular subtype. Genes differentially expressed between IBC and nIBC cells post 4 hours of TGFb1 revealed attenuated expression of SMAD3 transcriptional regulators with concomitant overexpression of MYC target genes in IBC. IBC patient samplesdemonstrated a near absence of nuclear SMAD3 expression in the primary tumors from (P < 0.001) and a further reduction of SMAD3 staining intensity was observed in tumor emboli (P ¼ 0.019). Integration of gene and protein expressiondata revealed that a substantial fraction of the IBC signature genes correlated with SMAD3 and these genes (i.e. 21/24; P < 0.001) are indicative of attenuated SMAD3 signaling in IBC. Conclusions: We demonstrate that IBC cells are characterized by attenuated SMAD3 protein expression and transcriptional activity, leading to obliteration of the cell motility inducing capacity of TGFb1. Recent studies revealed that SMAD3 is essential for TGFb1 induced cell motility through induction of epithelial to mesenchymal transition (EMT). This study suggests that in the absence of SMAD3 expression, a partial EMT is induced leading to collectively invading cancer cells that are gifted with a high metastatic potential, thereby providing an intriguing explanatory model of the biology of tumor emboli in IBC. Legal entity responsible for the study: University of Antwerp. Funding: Kom Op Tegen kanker -Emmanuel Van der Schueren.