In contrast, pathways such as mitotic roles of Polo-like kinase, Rac signaling, aryl hydrocarbon receptor signaling, GM-CSF signaling, and CD40 signaling were activated or had a trend towards activation in doxorubicin-treated MCF7/LMTK3 cells compared to MCF7 (Fig. a mouse model we showed that ectopic overexpression of LMTK3 decreases the efficacy of doxorubicin in reducing tumor growth. Interestingly, breast cancer cells overexpressing LMTK3 delayed the generation MF498 of double strand breaks (DSBs) after exposure to doxorubicin, as measured by the formation of H2AX foci. This effect was at least partly mediated by decreased activity of ataxia-telangiectasia mutated kinase (ATM) as indicated by its reduced phosphorylation levels. In addition, our RNA-seq analyses showed that doxorubicin differentially regulated the expression of over 700 genes depending on LMTK3 protein expression levels. Furthermore, these genes were MF498 found to promote DNA repair, cell viability and tumorigenesis processes / pathways in LMTK3-overexpressing MCF7 cells. In human cancers, immunohistochemistry staining of LMTK3 in pre- and MF498 post-chemotherapy breast tumor pairs from four separate clinical cohorts revealed a significant increase of LMTK3 following both doxorubicin and docetaxel based chemotherapy. In aggregate, our findings show for the first time a contribution of LMTK3 in cytotoxic drug resistance in breast cancer. Introduction Lemur tyrosine kinase 3 (and between MCF7 and MCF7/LMTK3 cells upon treatment with DMSO or 1?M doxorubicin Our Venn diagram also pointed to an intriguing result where doxorubicin differentially regulated MF498 the expression of SOX6 and HEY1 transcription factors in MCF7 and MCF7/LMTK3 cells (Fig. ?(Fig.3e).3e). In particular, doxorubicin suppressed the expression Itgax of SOX6 in MCF7 cells (~?6-fold), whereas it increased it in MCF7/LMTK3 cells (~4-fold). In contrast, doxorubicin potentiated the expression of HEY1 by ~3??fold in MCF7 cells and suppressed it by ~2??fold in MCF7/LMTK3 (Fig. ?(Fig.3e3e). To further inquire if there were additional genes that differentially responded to doxorubicin treatment between MCF7 and MCF7/LMTK3 cells (from now on labelled as: Dox:LMTK3 genes), we re-analyzed the RNA-Seq data using the interaction model provided by DESeq2. This model tests for genes that respond differently to doxorubicin treatment across MCF7 and MCF7/LMTK3 by controlling for differences between cell lines due to LMTK3 overexpression and doxorubicin treatment effect on MCF7 cells. The model identified that 896 genes responded differently (at the value, for significance in fold change, is plotted on the score) in doxorubicin-treated MCF7/LMTK3 cells compared to doxorubicin-treated MCF7 cells. In contrast, pathways such as mitotic roles of Polo-like kinase, Rac signaling, aryl hydrocarbon receptor signaling, GM-CSF signaling, and CD40 signaling were activated or had a trend towards activation in doxorubicin-treated MCF7/LMTK3 cells compared to MCF7 (Fig. ?(Fig.5a5a and Supplementary excel file 5). The IPA also revealed a significant decrease in doxorubicin-mediated inhibition of several biological functions including cell survival, cell viability of tumor cells and DNA repair, as well as, a significant decrease in doxorubicin-mediated activation of biological functions such as cell death of tumor cells, the formation of H2AX and chromosomal instability in doxorubicin-treated MCF7/LMTK3 cells compared to doxorubicin-treated MCF7 cells (Fig. ?(Fig.5b5b and Supplementary excel file 6). Open in a separate window Fig. 5 Functional analysis of doxorubicin-LMTK3 mediated differential gene expression. a Heatmaps comparing scores of canonical pathways significantly enriched for doxorubicin regulated genes identified from doxorubicin/DMSO treated MCF7 and MCF7/LMTK3 cells. The significant score. A score of 2 is considered as significant activation and a score between (0, 2) or (?2, 0) represents trend towards activation or inhibition, respectively. b A bar graph comparing scores of disease biological functions enriched for doxorubicin regulated genes identified from doxorubicin/DMSO treated MCF7 and MCF7/LMTK3 cells. c Functional classification of Dox:LMTK3 genes identified using PANTHER classification system. dCf GO pathways analysis of the protein-protein interaction clusters identified in Dox:LMTK3 genes using fast-greedy algorithm provided with STRING database. The STRING network analysis was then performed on gene products involved in RNA processing (d: Cluster 1), DNA repair (e: Cluster 2), and regulation of cell loss of life (f: Cluster 3). The blue, green and red colorization in STRING network of RNA digesting represents protein involved with splicesome, ribosome and ribosome biogenesis respectively. The reddish colored and blue color in STRING network of DNA restoration represents proteins involved with Nucleotide Excision Restoration and Foundation Excision Restoration. The red colorization in STRING network of rules of cell loss of life represents proteins involved with downregulation of apoptotic pathways. For all your STRING networks, the effectiveness of the dark line indicates power of the info support for confirmed protein-protein association Furthermore,.