Spinophilin a putative tumor suppressor gene has been shown to be involved in the pathogenesis of certain types of cancer but its role has never been systematically explored in breast cancer. AG14361 prognostic factor in BC patients (hazard ratio: 1.93 95 confidence interval 1.24-3.03; = 0.004). In addition to spinophilin levels age tumor stage and negative hormone receptor status were independent prognostic factors (<0.001 for all parameters). To further characterize the biological role of spinophilin expression in BC cells we used a shRNA lentiviral vector system to transduce and silence spinophilin. We selected SUM159 cells as a basal-like cell line model and MCF-7 cells as a luminal A cellular model [13]. SUM159 cells are p53 gene mutated whereas MCF-7 cells are p53 wild-type cells [14]. Analogous to the TGCA patient data SUM159 cells have naturally occurring lower spinophilin levels than MCF-7 cells in qRT-PCR and Western AG14361 Blot analysis (Supplementary Figure 1A and 1B). Using shRNA a silencing effect was confirmed by reduced spinophilin protein levels in Western Blot analyses for both cell lines (Supplementary Figure 2). Subsequently we explored the effects of reduced spinophilin expression on cellular growth rates of these cell lines. A significantly increased cellular growth could be detected in spinophilin silenced MCF-7 cells (78% AG14361 increase ±12% features < 0.05). Figure 4 metastases formation and gene expression profile in spinophilin-silenced basal-like breast cancer cells After identifying that low spinophilin expression is associated with aggressive biological behavior in BC cells we further tried to figure out AG14361 which genes are most differentially up- or down-regulated in spinophilin-silenced BC cells. Therefore we performed microarray gene expression analysis in three independent biological replicates comparing SUM159 spinophilin-silenced and control cells. Most important changes of gene expression are shown in the Heat map in Figure ?Figure4A4A and a list of the 30 top up- and down-regulated genes as well as a pathway analysis is included in Supplementary Table 2 and 3. Consequently Rabbit Polyclonal to SERPINB4. the five most up- and down-regulated protein-coding genes were further validated using quantitative RT-PCR. A 100% concordance between microarray results and the confirmatory RT-PCR was found. Under the differentially expressed genes we identified several genes previously related to cancer including the up-regulated and the down-regulated and (Figure 4B C). Using the expression data of the 921 BC patients of the TCGA dataset we confirmed for some of these differentially expressed genes including and < 0.05). DISCUSSION Spinophilin is a protein phosphatase 1 binding protein that AG14361 has been initially described in dendritic spines of the nervous system [7]. Previous studies reported that spinophilin is important for cell-cell adhesion and functions as a link between the actin cytoskeleton and the plasma membrane [15-17]. Basically discovered to be essential for several processes in the nervous system spinophilin has later been associated with certain types of cancer. Vivo and colleagues were the first who reported a connection between the human tumor suppressor protein ARF and spinophilin [18]. More recently published studies confirmed a role for spinophilin in human cancer. For instance spinophilin expression had an inhibitory effect on anchorage-independent growth of glioblastoma cells [18 19 as well as an effect on self-renewal and differentiation in brain tumor stem cells [20]. Molino-Pinelo and colleagues revealed that spinophilin expression correlates with higher grade of malignancy in lung cancers [9]. In hepatocellular carcinoma reduced levels of spinophilin have been associated with high proliferation and poor prognosis [10]. Another recent study suggests that down-regulation of spinophilin in colorectal cancer correlates with a more aggressive histologic phenotype faster relapse and poorer survival in advanced stages of colorectal carcinoma [12]. Ress et al. confirmed the role of spinophilin in colorectal cancer and showed that reduced spinophilin levels led to increased cellular growth rates anchorage-independent growth [21]. Interestingly abnormalities in the growth of mammalian ducts have been observed in spinophilin knock-out mice [7]. In the present study which is the first one investigating the role of spinophilin in BC we observed a membranous localization of spinophilin in.