High expression of the Notch ligand Jagged-1 is associated with poor prognosis after surgery for colorectal cancer

Abstract

The importance of Notch signaling in colorectal cancer (CRC) carcinogenesis and progression has previously been presented. Increased expression of Jagged-1 (JAG1), a Notch ligand, in CRC has been revealed, but the detailed prognostic significance of JAG1 in CRC has not been determined. Protein expression of JAG1 was examined using immunohistochemistry in 158 CRC specimens. Expression of JAG1 and E-cadherin and their associations with clinicopathologic characteristics, overall survival (OS) and relapse-free survival (RFS) were evaluated. In vitro studies using compounds to regulate intracellular signaling and small interfering RNA to silence JAG1 were performed in a colon cancer cell line. JAG1 expression in cancerous tissues was weak, moderate or strong in 32%, 36% and 32% of specimens, respectively, and correlated with histologic type and T stage. In multivariate analysis, JAG1 expression, histologic type and lymphatic invasion independently correlated with OS and RFS. The combination of high JAG1 expression and low E-cadherin expression had an additive effect toward poorer OS and RFS compared with the low JAG1/high E-cadherin expression subtype. A significant correlation between JAG1 expression and KRAS status was detected in groups stratified as high E-cadherin expression. In vitro studies suggested that RAS-MEK-MAP kinase and the Wnt pathways positively regulated JAG1 expression. Gene silencing with siJAG1 indicated that JAG1 promotes the transition from epithelial to mesenchymal characteristics and cell growth. High expression of JAG1 is regulated by various pathways and is associated with poor prognosis through promoting the epithelial to mesenchymal transition and cell proliferation or maintaining cell survival in CRC.

Keywords: Colorectal cancer; JAG1; Notch; epithelial-mesenchymal transition; survival rate.

Figure 1

Representative immunohistochemical staining of JAG1 expression in human colorectal cancer tissues (original magnification ×100, scale bars represent 0.25 mm). (a) Example of cancer and endothelium tissue with weak intensity of staining (jcIHC‐W, jeIHC‐W). (b) Example of cancer and endothelium with moderate intensity of staining (jcIHC‐M, jeIHC‐M). (c) Example of cancer and endothelium with moderate and strong intensity of staining, respectively (jcIHC‐M, jeIHC‐S). (d) Example of cancer and endothelium with strong intensity of staining (jcIHC‐S, jeIHC‐S). (e) Example of poorly differentiated carcinoma with a strong intensity of staining. Representative each five regions in cancer or endothelium were indicated by open or filled arrow‐heads, respectively.

Figure 2

Prognostic significance of JAG1 expression in cancer cells by analysis of Kaplan–Meier estimates. (a) Kaplan–Meier estimates of 10‐year overall survival (OS) in all CRC patients and (b) 5‐year recurrence‐free survival (RFS) in patients except for Stage IV CRC according to staining intensity. Mod indicates moderate.

Figure 3

Prognostic significance of JAG1 expression in cancer cells stratified by E‐cadherin expression (based on intensity of staining) shown by analysis of Kaplan–Meier estimates and Cox proportional hazards model. (a, b) Kaplan–Meier estimate of 5‐year OS (a) and 5‐year RFS (b) in CRC patients according to staining intensity of JAG1 expression in cancer cells stratified by E‐cadherin expression. jcIHC‐W, ‐M, ‐S indicate weak, moderate, and strong intensity of staining of JAG1 expression in cancer cells, respectively. eIHC‐In2/3 and eIHC‐In1 indicate staining intensity of 2/3 and 1 for E‐cadherin expression, respectively. (c) 3‐year survival rate calculated by analysis of the Kaplan–Meier estimates shown in (a) and (b). (d) Hazard ratio (HR) and 95 % confidence interval (CI) of JAG1 and E‐cadherin expression analyzed by Cox proportional hazards model versus jcIHC‐W/eIHC‐In2/3 group.

Figure 4

Mechanisms of regulation of JAG1 expression based on in vitro study. (a) The effect of inhibition of RAS‐MEK‐MAP kinase pathway on JAG1 expression and induction of EMT‐like phenotype was examined using the MEK inhibitor PD325901 in the colon cancer cell line HCT‐116. In the left panel, blue staining indicates the nucleus and green staining indicates JAG1 protein. In the middle panel, blue staining indicates the nucleus and red staining indicates E‐cadherin protein. In the right panel, blue, green, and red staining indicate the nucleus, SNAIL, and JAG1 protein, respectively. Scale bars represent 50 µm. (b) Fluorescence intensity of JAG1, Snail, and E‐cadherin was analyzed. Data are presented as mean ± SD of 20 fields of view. *, P < 0.05, Student's t‐test. (c) Effects of GSK3β inhibitor on JAG1 protein expression and EMT‐like phenotype. JAG1, E‐cadherin, and SNAIL protein expression was analyzed by western blotting.

Figure 5

Effect of JAG1 gene silencing on proliferation and epithelial mesenchymal transition (EMT)‐like phenotype based on in vitro study. (a, b) Effect of small interfering RNA for JAG1 (siJAG1) on JAG1 expression and EMT‐like phenotype. E‐cadherin and SNAIL protein expression (a) or E‐cadherin (CDH1) and vimentin (VIM) mRNA expression (b) were analyzed by western blotting and qRT‐PCR respectively. Non‐targeting siRNA (siNON) was used as a negative control. Expression levels of mRNA are indicated relative to expression with siNON treatment. Data are mean ± SD of nine wells. (c) Effect of siJAG1 on JAG1 and HES1 mRNA expression in p53 ^−/− and wild type (Wt) HCT‐116 cells. mRNA expression was presented as a ratio relative to expression in Wt cells treated with siNON in the left two panels. JAG1 mRNA expression in Wt or p53 ^−/− cells treated with siJAG1 was also presented as a ratio relative to expression in Wt or p53‐/‐ cells treated with siNON in the right panels. Data are mean ± SD of nine wells. (d) Effect of siJAG1 on JAG1 protein expression in p53 ^−/− and Wt cells analyzed by western blotting. (e) Effect of siJAG1 on cell growth in p53 ^−/− and Wt cells. siJAG1 treatment was initiated 2 days after plating. Data are presented as mean ± SD of six wells for each time point (result from three independent experiments). Statistical analysis was performed by Student's t‐test. * or ^#, P < 0.05.

Figure 6

Hypothesized mechanisms of cancer recurrence or death induced by JAG1‐Notch pathway activation following increased JAG1 expression regulated by various factors.