The CaSm (LSm1) oncogene promotes transformation, chemoresistance and metastasis of pancreatic cancer cells

Abstract

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. Evidence indicates that CaSm modulates messenger RNA degradation; however, its target genes and the mechanisms by which CaSm promotes pancreatic cancer remain largely unknown. Here, we demonstrate that the CaSm overexpression alters several hallmarks of cancer-including transformation, proliferation, chemoresistance and metastasis. Doxycycline-induced CaSm expression enhanced proliferation and both anchorage-dependent and -independent growth of the human Panc-1 cells in vitro. CaSm induction decreased gemcitabine-induced cytotoxicity and altered the expression of apoptotic regulation genes, including Bad, E2F1 and Bcl-XL. CaSm-overexpressing Panc-1 cells were twofold more migratory and fourfold more invasive than the driver controls and demonstrated characteristics of epithelial-to-mesenchymal transition such as morphological changes and decreased E-cadherin expression. CaSm induction resulted in changes in RNA expression of metastasis-associated genes such as MMP1, SerpinB5, uPAR and Slug. Using a murine model of metastatic pancreatic cancer, injection of CaSm-induced Panc-1 cells resulted in a higher abundance of hepatic metastatic lesions. Overall, CaSm overexpression contributed to a more aggressive cancer phenotype in Panc-1 cells, further supporting the use of CaSm as a therapeutic target against pancreatic cancer.

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 absence or presence of 1 μg/ml doxycycline for 24 h before assessed for CaSm expression by western blot analysis with GAPDH as a control. (c) In all, 100 000 tet-on driver and tet-on CaSm Panc-1 cells were plated in triplicate in six-well plates on day 0. Cell proliferation was quantified with Trypan blue cell counts on days 1, 3 and 4. Depicted is a representative of three independent experiments with mean cell count and s.e.m. shown. (d) Tet-on driver and tet-on CaSm Panc-1 cells were plated at 50 cells per well or 100 cells per well in triplicate in six-well plates with media containing 1 μg/ml doxycycline. After 2 weeks, cells were stained with 0.01% crystal violet and colonies containing at least 50 cells quantified. The mean quantification and s.e.m. are depicted. (e) Tet-on driver and tet-on CaSm cells were plated at 500 or 1000 cells per well in 0.4% agar in six-well plates. In all, 0.4% agar containing growth media and 1 μg/ml doxycycline was added once a week. After 5 weeks, cells were stained with 0.1% crystal violet in 10% ethanol and quantified by counting the number of resulting colonies at least 0.1 mm in diameter. Depicted is a representative of three independent experiments with mean cell count and s.e.m. shown. *P<0.05, ***P<0.001.

Figure 2

CaSm overexpression protects Panc-1 cells from gemcitabine-induced cytotoxicity. (a, b) Tet-on driver and tet-on CaSm Panc-1 cells were grown in 1 μg/ml doxycycline before being treated with 200 nM gemcitabine on day 0. Cells were trypsinized and fixed in ice-cold ethanol on days 0, 1, 3 and 5, stained with 0.1 μg/μl PI and analyzed for cell cycle content by flow cytometry analysis. (a) Cell cycle distribution on days 0 and 5, (b) depicts quantification of mean sub-G1 content on individual days along with s.e.m., (c) Tet-on driver or CaSm Panc-1 cells were seeded in each well of a 96-well plate (5000 cells per well) overnight. Cells were then treated with 0–100 μM gemcitabine with replicates of at least six wells per condition. Ninety-six hours after treatment, cells were fixed with 5% trichloroacetic acid, stained with 0.4% SRB, and dissolved in 10 mM Tris base before absorbance was read at 560 nm. Depicted is a representative of three independent experiments, along with nonlinear regression (y=linear, x=logarithmic) and respective R² values. *P<0.05, **P<0.005, ***P<0.001.

Figure 3

CaSm induction alters apoptotic gene expression. (a) Tet-on driver and tet-on CaSm Panc-1 cells were maintained in 1 μg/ml doxycycline for 4 weeks before RNA expression was analyzed by the SABioscience Cancer PathwayFinder PCR-based array. Genes with a 1.5-fold or greater alteration comparing means of two experimental arrays with two driver+dox controls are depicted. (b) Real-time PCR was used to validate the microarray with GAPDH used as a control. ***P<0.001.

Figure 4

CaSm induction promotes migration and invasion and alters metastatic gene expression. Tet-on driver and tet-on CaSm Panc-1 cells, previously grown in 1 μg/ml doxycycline for 4–8 weeks, were plated in serum-free DMEM onto the top chamber of fibronectin-coated transwell membranes (a) or rehydrated Matrigel transwell chambers (b). DMEM supplemented with 10% FBS was used as the chemoattractant in the bottom wells. The chambers were incubated at 37 °C for 4 h (a) and 48 h (b) before transwell cells were stained and quantified by counting random fields at × 40 magnification. Represented is mean and s.e.m. (c) Real-time PCR was performed to validate the microarray with samples normalized to GAPDH as a loading control. *P<0.05, ***P<0.001; NS, not significant.

Figure 5

Overexpression of CaSm promotes EMT transition. (a) Tet-on driver and tet-on CaSm cells were grown in 1 μg/ml doxycycycline. Images were captured at × 100 magnification by Qcapture pro software (Surrey, BC, Canada). (b) E- and N-cadherin expression was assessed using whole-cell lysates from tet-on driver and tet-on CaSm Panc-1 cells separated on an 8% polyacrylamide gel with GAPDH used as loading control. (c) A panel of EMT transcription factors was evaluated by real-time PCR comparing expression between tet-on driver and tet-on CaSm Panc-1 cells grown under chronic doxycycline culture conditions (1 μg/ml). (d) Tet-on driver and tet-on CaSm cells were maintained with 1 μg/ml doxycycline before tet-on CaSm cells were either left untreated or transfected with 5 μg Slug or Scramble siRNA using Lipofectamine RNAi Max reagent. Cells were harvested 72 h after transfection for RNA isolation and real-time PCR (left) or plated for 4-h migration assay as previously described (right). Shown is a representative of three independent experiments with mean and s.e.m. depicted. **P<0.005, ***P<0.001, NS, not significant (P>0.05).

Figure 6

Induced CaSm overexpression enhances tumor formation and metastasis in NSG mice. (a) Tet-on driver and tet-on CaSm Panc-1 cells were maintained in chronic doxycycline (1 μg/ml) for 2–3 weeks before to subcutaneous injection of 0.1 × 10⁶ (n=10 for both tet-on driver and tet-on CaSm) or 2 × 10⁶ (n=9 tet-on driver and n=10 tet-on CaSm) cells on day 0. Expression was induced with 625 mg/kg doxycycline feed. Tumor presence was evaluated twice weekly by palpation beginning at day 7. Depicted is the accumulation of two independent experiments. (b–d) Tet-on driver and tet-on CaSm Panc-1 cells were maintained in chronic doxycycline (1 μg/ml) for 3 weeks before splenic injection on day 0 (n=6 driver, and n=6 CaSm). Expression was induced with 625 mg/kg doxycycline feed and animals were weighed weekly starting 3 weeks post-injection. After 6 weeks, animals were killed and livers and spleens were resected. Liver tissue was fixed in 10% formalin overnight and sectioned (one section cut every 500 μm, for five total sections per animal). Sections were stained for hematoxylin and eosin (H&E) (representative images, b) and assessed for the number of metastatic lesions/100 mm² total tissue (c) by ImageJ, with at least five images per section assessed at × 40 magnification. Lesions with a diameter of at least 0.5 mm were quantified. (d) Animal weight was measured weekly beginning at week 3 post-injection. Shown here is the average weight loss compared with individual pre-injection weight. *P<0.05, **P<0.005, ***P<0.001.