High-level expression of Notch1 increased the risk of metastasis in T1 stage clear cell renal cell carcinoma

Abstract

Background: Although metastasis of clear cell renal cell carcinoma (ccRCC) is basically observed in late stage tumors, T1 stage metastasis of ccRCC can also be found with no definite molecular cause resulting inappropriate selection of surgery method and poor prognosis. Notch signaling is a conserved, widely expressed signal pathway that mediates various cellular processes in normal development and tumorigenesis. This study aims to explore the potential role and mechanism of Notch signaling in the metastasis of T1 stage ccRCC.

Methodology/principal findings: The expression of Notch1 and Jagged1 were analyzed in tumor tissues and matched normal adjacent tissues obtained from 51 ccRCC patients. Compared to non-tumor tissues, Notch1 and Jagged1 expression was significantly elevated both in mRNA and protein levels in tumors. Tissue samples of localized and metastatic tumors were divided into three groups based on their tumor stages and the relative mRNA expression of Notch1 and Jagged1 were analyzed. Compared to localized tumors, Notch1 expression was significantly elevated in metastatic tumors in T1 stage while Jagged1 expression was not statistically different between localized and metastatic tumors of all stages. The average size of metastatic tumors was significantly larger than localized tumors in T1 stage ccRCC and the elevated expression of Notch1 was significantly positive correlated with the tumor diameter. The functional significance of Notch signaling was studied by transfection of 786-O, Caki-1 and HKC cell lines with full-length expression plasmids of Notch1 and Jagged1. Compared to the corresponding controls, all cell lines demonstrated significant promotion in cell proliferation and migration while cell cycle remained unaffected.

Conclusions/significance: High-level expression of Notch signaling increased the risk of metastasis in T1 stage ccRCC by stimulating the proliferation and migration of tumor cells, which may be helpful for the selection of suitable operation method and prognosis of ccRCC.

Figure 1. Notch1 and Jagged1 expression in ccRCC tissues.

(A) mRNA expression detected by real-time RT-PCR showing elevated Notch1 mRNA expression in localized and metastatic tumors compared to non-tumor(NT) tissues (P = 0.001 and P = 0.000 respectively), and higher expression in metastatic tumors compared to localized tumors (P = 0.028); On the right panel, analysis showing elevated Jagged1 mRNA expression in tumors compared to NT (P = 0.000 and P = 0.000 for localized and metastatic tumors respectively), but lower expression in metastatic tumors compared to localized tumors (P = 0.005). Each dot representing a tissue sample. (B) Protein expression detected by western-blot assay showing elevated expression of Notch1 and Jagged1 protein in tumor tissues compared to non-tumor tissues. The right panel was the densitometric analysis of the bands. The data shown are mean±SD.

Figure 2. Notch1 and Jagged1 mRNA expression analysis at different stages in ccRCC samples.

(A) Analysis showing significantly higher expression of Notch1 in metastatic tumors at T1 stage compared to localized tumors(P = 0.001). (B) No statistically significant difference of Jagged1 expression in all three stages. The data shown are mean±SD.

Figure 3. Analysis of tumor diameter and Notch1 and Jagged1 mRNA expression level in T1 stage ccRCC.

(A) Analysis showing larger average diameter in metastatic tumors (6.375±0.479 cm n = 4) compared to localized tumors (4.089±1.237 cm, n = 19, P = 0.025). (B) Positive correlation of Notch1 expression and tumor diameter in T1 stage(n = 23, R = 0.435, P = 0.038). (C) No correlation of Jagged1 expression and tumor size in T1 stage(n = 23, R = −0.172, P = 0.432). The data shown are mean±SD.

Figure 4. Notch1 promoting the proliferation in tumor and normal kidney cell lines.

Proliferation assay by MTS showing increased proliferation in Caki-1 and 786-O cell line after Notch1 and Jagged1 expression. MTS assay in HKC cell line showing increased proliferation rate in Notch1 expressing cells only. The data shown are means±SD from two independent experiments, each carried out in triplicate.

Figure 5. Notch1 and Jagged1 promoting the migration in tumor cell lines.

(A)–(C) are representative view of 786-O cell line transfected with Notch1, Jagged1 and control plasmid respectively. (D)–(F) are Caki-1 cell line transfected with Notch1, Jagged1 and control plasmid respectively. (G) Notch1 and Jagged1 can promote the migration of 786-O and Caki-1 tumor cell lines compared to the controls. (H) MMP-9 mRNA expression was significantly elevated in Caki-1 and 786-O cell lines after transfected with Notch1 and Jagged1 plasmid. The data shown are mean±SD.

Figure 6. Analysis of cell cycle with over-expression of Notch1 and Jagged1.

(A)–(B) In 786-O, Caki-1 and HKC cell line, cell cycle analysis demonstrating no difference between cells over-expressing Notch1 and Jagged1 compared to controls. (C) P21 and P27 remained unchanged in all cell lines after transfected with Notch1 and Jagged1 plasmids.