The Notch signaling pathway is related to neurovascular progression of pancreatic cancer

Abstract

Objective: To analyze the potential role of the Notch signaling pathway in pancreatic cancer angiogenesis and invasion.

Background: Angiogenesis, pain, and early neuroinvasion are clinical features of pancreatic cancer. Blood vessels and nerves develop together and use common routes through the organism. The Notch pathway (Notch-1/4, Jagged-1/2, Delta-1) appears crucial in this process. The current study analyzed the Notch pathway in pancreatic cancer and characterized its angiogenic and invasive effects.

Methods: Five PaCa cell lines were cultured for the in vitro experiments. Real-time quantitative RT-PCR was done to quantify mRNA expression in 31 human PaCa specimens, and immunohistochemistry was used to localize protein expression within tumor specimens. Activation of the Notch signaling was done by transfection of PaCa cells with a constitutive active Notch-1 mutant (Notch-IC). Overexpression of Jagged and Delta was achieved by transfection of full-length cDNA. Spheroid assays were used to study angiogenesis and ELISAs to measure VEGF, bFGF, and angiogenin expression. Matrigel invasion assays were used to analyze tumor cell invasion.

Results: Notch-3 and Notch-4 mRNA were significantly (P < 0.001) overexpressed in PaCa. Immunohistochemistry revealed protein accumulation of Notch-1 as well. All ligands were significantly up-regulated. A positive immunosignal of ligands was seen in nerves, blood vessels, and ductal tumor cells. Transfection of PaCa cells with the constitutive active Notch-IC mutant and with Jagged-1 revealed increased levels for VEGF. Concomitantly, recombinant Jagged-1 increased sprouting of endothelial cells in the spheroid assay.

Conclusion: The Notch pathway most likely regulates neurovascular development in pancreatic cancer. Activation of this signaling pathway by constitutive Notch-1 mutants and by Jagged-1 causes an angiogenic and invasive tumor phenotype. Specific blockade of Notch signaling may therefore be beneficial for patients with pancreatic cancer.

FIGURE 1. Real-time quantitative RT-PCR analysis: Expression of Notch-1, Notch-2, Notch-3, Notch-4, Jagged-1, Jagged-2, and Delta was quantified in 6 human pancreatic cancer cell lines (A–F). Notch-2 was detectable in all pancreatic cancer cell lines, whereas other members of the Notch gene family were differentially expressed. Among the ligands, Jagged-1 was expressed in all cell lines, whereas Jagged-2 and Delta-1 mRNA expression was present in some. Normalization of expression levels was done using cyclophilin-B as a housekeeping gene. *P < 0.001. #P = 0.0593.

FIGURE 2. Real-time quantitative RT-PCR: human specimens. Expression of Notch-1, Notch-2, Notch-3, Notch-4, Jagged-1, Jagged-2, and Delta was quantified in 31 human pancreatic cancer specimens (black bar) and compared with expression of these mRNA moieties in normal pancreatic tissue specimens (clear bar) (n = 22). Notch-3 and Notch-4 were statistically significantly overexpressed (*P < 0.001), whereas differences in Notch-2 mRNA expression did not reach statistical significance (#P = 0.0593). The ligands Jagged-1 and -2 as well as Delta were significantly overexpressed in pancreatic cancer specimens (*P < 0.001).

FIGURE 3. Immunohistochemistry of Notch family members in pancreatic cancer samples. Notch-1 immunoreactivity was present in ductal pancreatic cancer cells (A; black arrow) but also in intratumoral nerves (B; white arrow), especially when nerves were infiltrated by cancer cells (C, D). Notch-2 was detectable in tumor cells (E, F) and also in nerves. Please note the nuclear staining (activated Notch) in ductal cancer cells (G). Notch-3 immunoreactivity was marked by staining in ductal pancreatic cancer cells but also in vascular smooth muscle cells (I, J; gray arrow). Tumor cells stained positive for Notch-3 (H), but periendothelial vascular smooth muscle cells also exhibited Notch-3 immunoreactivity (I, J). Notch-4 was found in tumor cells but also in endothelial linings (K, L; red arrow).

FIGURE 4. Immunohistochemistry of Notch ligands in pancreatic cancer tissues. Strong immunoreactivity was present for Jagged-1 in ductal cancer cells (A; black arrow), in so-called “tubular complexes” (B), but also in intratumoral nerves (B; white arrow), especially when they were infiltrated by cancer cells (C, D). Furthermore, a strong signal was detectable in the islets of Langerhans (E, F). Cytoplasmic Delta-1 immunoreactivity was found in cancer cells (G, H) but also in vascular smooth muscle cells, whereas the endothelial linings remained negative (I, red arrow).

FIGURE 5. Overexpression of Notch-1 IC, Jagged-1, and Delta-1 in cultured pancreatic cancer cells: Transfection studies in pancreatic cancer cells were performed as described in Materials and Methods. Angiogenin, VEGF, and basic FGF were measured by specific ELISAs. Overexpression of Jagged-1 resulted in increased VEGF levels in the supernatant of pancreatic cancer cells (A). Similarly, expression of a constitutive active Notch variant (Notch-1 IC) also increased VEGF levels in both tested cell lines. Delta-1 overexpression did not result in changes in the VEGF levels. Basic FGF secretion was higher in the case of Notch-1-IC expression in the MIA PaCa-2 cell line but not in the AsPC-1 cell line (B). Neither Jagged-1 nor Delta-1 transfection changed basic FGF protein expression (B). Low levels of angiogenin in MIA PaCa-2 cells were not changed by Jagged-1 or Delta-1 transfection. A high constitutive expression level of angiogenin was detectable in the AsPC-1 cell line. Upon transfection of this cell line with Delta-1, angiogenin expression increased further. No changes in the expression levels of angiogenin were seen upon transfection with Jagged-1 or Notch-1 IC. *P < 0.05.

FIGURE 6. Three-dimensional endothelial cell sprouting assay: Endothelial cell spheroids were seeded as described. Basal sprouting activity is shown in A. Addition of VEGF (25 ng) resulted in vigorous sprouting (B) of HUVECs into this matrix. D, E, Increasing doses (25, 50, and 250 ng/mL) of recombinant Jagged-1 protein were added to HUVEC spheroids. The highest tested dose of 250 ng exhibited an angiogenic stimulus similar to that of VEGF at a dose of 25 ng. The cumulative increase under various growth conditions is summarized in F. *P < 0.05.

FIGURE 7. Matrigel Invasion Assays: The rat pancreatic cancer cell line DSL-6A/C1 was used in the Matrigel invasion assays. In each well, 5 × 10³ cells were seeded. Recombinant rat Jagged protein was added in the indicated doses. The invasion assay was analyzed after 24 hours. Invaded cells adhered to the lower surface were counted as described in Materials and Methods. * P < 0.05.