The cancer-associated Sm-like (CaSm) oncogene is overexpressed in 87% of human

The cancer-associated Sm-like (CaSm) oncogene is overexpressed in 87% of human pancreatic tumor samples and CaSm knockdown has demonstrated therapeutic efficacy in murine models of pancreatic cancer. cancer that has had few new therapies developed in the past decade. Beyond the LSm1 correlates relatively little remains known about the role for CaSm in the process of neoplastic transformation or how its downregulation in the setting of established neoplasia leads to decreased tumor RO5126766 growth. To explore this further we developed models that would allow the induction of CaSm overexpression in a PC cell line intrinsically expressing relatively low CaSm levels. Here we demonstrate that induced CaSm expression results in increased growth decreased chemotherapeutic sensitivity and enhanced migration/invasion BMP2 in PC cells. CaSm upregulation alters the gene expression of critical mediators of apoptosis metastasis and epithelial-to-mesenchymal transition (EMT) which complements the proposed function for CaSm in mRNA regulation and provides a putative mechanism for CaSm-mediated neoplastic progression. Results Induction of CaSm levels in human Panc-1 cells CaSm protein expression was evaluated via western RO5126766 blot analysis in a panel of human PC cell lines to identify a cell line with lower levels of basal expression. CaSm expression was variable among the cell lines with Panc-1 cells demonstrating the lowest endogenous levels (Figure 1a). Owing to their low endogenous CaSm expression Panc-1 cells were selected for gain-of-function studies to better identify the molecular consequences of CaSm upregulation. Panc-1 cells were stably transfected with the Clontech RetroX system to create a doxycycline-induced CaSm pool (tet-on CaSm) with tet-on driver Panc-1 cells as a control (Figure 1b). CaSm expression was induced in a doxycycline dose-dependent manner (Supplementary Figure 1a) with greatest induction achieved with 1?μg/ml doxycycline which was used in all subsequent experiments. Induced CaSm expression reached maximum levels 8-12?h after addition of 1 1?μg/ml doxycycline (Supplementary Figure 1b). Figure 1 CaSm induction enhances the proliferation and transformation of Panc-1 cells. (a) CaSm expression was analyzed in human PC cell lines using western blot analysis with GAPDH as a control. (b) Tet-on driver and tet-on CaSm Panc-1 cells were grown in the … Induced CaSm overexpression promotes growth and transformation in Panc-1 cells Previous studies demonstrated that adenoviral-delivered antisense CaSm RNA reduced Panc-1 cell growth by 44%.18 Reciprocally induced CaSm expression significantly enhanced cellular growth twofold compared with driver and non-induced controls (Figure 1c). Low-density clonogenic assays identified that CaSm-overexpressing cells formed colonies more readily compared with driver controls. At 100 cells per well induced CaSm expression resulted in a nearly threefold increase in colony count (66±2.6 colonies in the tet-on CaSm Panc-1 compared with 23±4.4 colonies in the driver controls; Figure 1d). These differences were even more pronounced at lower plating densities of 50 cells per well where CaSm-induced cells produced four times the number of colonies (42±8.7 compared with 11±4; Figure 1d) indicating that CaSm produced a transformed cellular phenotype with less dependence on cellular density and cytokine interactions. RO5126766 Furthermore induced CaSm upregulation enhanced the Panc-1 colony formation in soft agar. After 5 weeks of incubation in 0.4% soft agar the tet-on CaSm Panc-1 cells established twice the number of colonies than the driver control cells at both 500 and 1000 cells per well (Figure 1e) providing further evidence that CaSm has a significant RO5126766 role in transformation and cancer development. CaSm induction results in chemoresistance to gemcitabine and tumor-initiating capability. Immunocompromised NOD Scid Gamma (NSG) mice received subcutaneous dorsal injections of 1 1 × 105 and 2 × 106 tet-on driver or tet-on CaSm cells and tumor presence was examined biweekly for 8 weeks by palpation. Although nearly all of the mice established tumors during the course of the study the tet-on CaSm cells formed tumors more readily at both cell concentrations (Figure 6a) demonstrating that induced CaSm expression was associated with enhanced tumor formation in these mice. By day 7 100 of mice injected with 2 million tet-on CaSm Panc-1 cells formed palpable tumors however complete tumor uptake of the tet-on driver Panc-1 cells did not occur until day.