Activation of Notch Signaling Is Required for Cholangiocarcinoma Progression and Is Enhanced by Inactivation of p53 <i>In Vivo</i>

<div><p>Cholangiocacinoma (CC) is a cancer disease with rising incidence. Notch signaling has been shown to be deregulated in many cancers. However, the role of this signaling pathway in the carcinogenesis of CC is still not fully explored. In this study, we investigated the effects of Notch inhibition by γ-secretase inhibitor IX (GSI IX) in cultured human CC cell lines and we established a transgenic mouse model with liver specific expression of the intracellular domain of Notch (Notch-ICD) and inactivation of tumor suppressor p53. GSI IX treatment effectively impaired cell proliferation, migration, invasion, epithelial to mesenchymal transition and growth of softagar colonies. <i>In vivo</i> overexpression of Notch-ICD together with an inactivation of p53 significantly increased tumor burden and showed CC characteristics. <i>Conclusion</i>: Our study highlights the importance of Notch signaling in the tumorigenesis of CC and demonstrates that additional inactivation of p53 <i>in vivo</i>.</p></div

GSI IX attenuate invasion of human cholangiocarcinoma cells.

<p>SZ1 (A) and TFK1 (A) cell lines were treated for 48 h with control (DMSO) and GSI (5 µM, 20 µM, 40 µM) to investigate the effect of GSI on invasiveness of human cholangiocarcinoma cell lines. The number of cells that invaded through the membrane was determined by light microscope (20X magnification) counterstained and invasion index (B,C) was calculated as described in Material and Methods and plotted in bar graphs. Both TFK1 and SZ1 showed significant decrease in number of invading cells by light microscope. P values were calculated with ANOVA analysis of variance along with Bonferroni post test. The error bar represents standard deviation. Differences were considered as statistically significant (*) when the P-value was less <0.05.</p

Summary of observation in NotchICD :: AlbCre :: p53 L/L mice.

<p>Summary of observation in NotchICD :: AlbCre :: p53 L/L mice.</p

NICD overexpression and loss of p53 is influencing the tumor development of cholangiocarcinomas in mice.

<p>Macro- und microscopic pictures of mice with expression of AlbCre and Notch-ICD and inactivation of p53 at different timepoints like indicated. Each mouse was dissected at the indicated timepoint. H&E staining (20X magnification) was performed and analysis was accordingly done in collaboration with an independent pathologist. Note, there was no tumor either macroscopically or microscopically detected with an age of 3 month. Starting with an age of 6 month, mice with expression of AlbCre, Notch-ICD and loss of p53 developed cholangiocarcinomas.</p

GSI IX inhibits cell proliferation in human cholangiocarcinoma cell lines.

<p>TFK1, SZ1 and EGI1 cells showed a different expression of the Notch downstream target Hes1 (A). The cell proliferation of (B) SZ1 and (C) TFK1 cells was measured by cell proliferation assay, GSI (5 µM, 20 µM, 40 µM) inhibited cell proliferation in a dose- and time-dependent manner. Light microscopic pictures (10X magnification) were taken at 96 h to show the effect of GSI on cell proliferation of (D) SZ1 and (E) TFK1. Note that these results reveal the anti-proliferative effects of GSI IX on human cholangiocarcinoma cells.</p

Notch plays a pivotal role for the regulation of migration in human cholangiocarcinoma cells.

<p>Treatment with GSI IX suppresses the migration potential of human cholangiocarcinoma cell lines SZ1 and TFK1. Wound healing experiments of (A) SZ1 and (C) TFK cells cultured with GSI (5 µM, 20 µM, 40 µM) or control (DMSO). A scratch was made at (time 0 h) in both SZ1 and TFK1 and maintained for 24 h in conditioned medium with GSI or DMSO. The dotted lines are representing the edges of the wound. Photographs were taken under light microscope (10X magnification). After 24 h (A) SZ1 showed significant inhibition under 5–40 µM GSI and (C) TFK1 with a dose of 20–40 µM GSI treatment. In DMSO treated cells 80% to 90% of the wound healing was observed after 24 hrs. (A,C) The migration index (B,D) was calculated as described in Material and Methods and plotted in bar graphs. P values were calculated with ANOVA analysis of variance along with Bonferroni post test. The error bar represents standard deviation. Differences were considered as statistically significant (*) when the P-value was less <0.05.</p

GSI treatment in sorted pancreatic tumor initiating CD44+/EpCAM+ cells reduces cell proliferation and selectively inhibits EMT.

<p>(A) Table showing the expression of CD44+, EpCAM+ cells and the combination of CD44+/EpCAM+ cells in pancreatic cancer cell line KP3. (B) CD44+/EpCAM+ cells were treated with GSI (2.5 µM, 5 µM and 10 µM) and control (DMSO) for 48 h to determine the role of Notch in regulating the cell proliferation. Cell proliferation was inhibited in a dose- and time-dependent manner. Note that these results reveal the anti-proliferative effects of GSI on human pancreatic tumor CD44+/EpCAM+ initiating cells. (C) Light microscopic pictures (10× magnification) were taken at 48 h to show the effect of GSI on cell proliferation. (D) The down regulation of the Notch pathway was confirmed by Western Blot for Notch downstream target Hes1. Compared to unsorted and sorted DMSO treated cells Hes1 showed a dose-dependent down regulation after GSI treatment. (D) CD44 and EpCAM were down regulated in a dose dependent manner. The black arrow is marking the protein lane of CD44. (E) Epithelial marker E-cadherin was unaltered, but mesenchymal marker N-cadherin, Vimentin and Slug showed dose-dependent down regulation.</p

Change in the expression of epithelial and mesenchymal cell markers after GSI IX treatment in human pancreatic cancer.

<p>(A) KP3 and (B) BxPC3 cells were treated with control (DMSO) and GSI (2.5 µM, 5 µM and 10 µM) for 96 h. The expression of EMT markers: E-cadherin, N-cadherin, Slug and Vimentin were analyzed by Western blot. β-actin was used as a loading control. Both (A) Kp-3 and (B) BxPC3 showed no change in expression of epithelial marker E-cadherin but resulted in a GSI dose-independent down regulation of mesenchymal markers N-cadherin and Vimentin. We also detected a down regulation of the EMT transcriptional factor Slug after GSI treatment for both pancreatic cancer cell lines.</p

GSI IX attenuate invasion of human pancreatic cancer cells.

<p>KP3 and BxPC3 cell lines were treated for 48 h with control (DMSO) and GSI (2.5 µM, 5 µM, 10 µM) to investigate the effect of GSI on invasiveness of pancreatic cancer cell lines. The number of cells that invaded through the membrane was determined by light microscope (20× magnification) counterstained and invasion index was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046514#s2" target="_blank">Material and Methods</a> and plotted in bar graphs. Both (A) KP3 and (B) BxPC3 showed significant decrease in number of invading cells by light microscope. Note the slight difference of invasion index between (C) Kp3 and (D) BxPC3 cells. P values are calculated with ANOVA analysis of variance along with Bonferroni post test. The error bar represents standard deviation. Differences were considered as statistically significant when the P-value was less <0.05 and non significant “n.s.” when the P-value was higher >0.05. The error bar represents standard deviation.</p

GSI IX inhibits cell proliferation in human pancreatic cancer cell lines and down regulates the Notch pathway downstream target Hes1.

<p>(A) KP3 and (B) BxPC3 cells were treated with GSI (2.5 µM, 5 µM, 10 µM) and control (DMSO) for 96 hrs showed a down regulation of Hes1 protein by Western Blot analysis. GSI treatment resulted in a shift in the growth curves. The cell proliferation of (C) KP3 and (D) BxPC3 was measured by cell proliferation assay, GSI inhibited cell proliferation in a dose- and time-dependent manner. Note that these results reveal the anti-proliferative effects of GSI on human pancreatic cancer cells. Light microscopic pictures (10× magnification) were taken at 96 h to show the effect of GSI on cell proliferation of (E) KP3 and (F) BxPC3.</p