The neurotrophic factor artemin promotes pancreatic cancer invasion

Abstract

Objective: To analyze the role of Artemin in pancreatic ductal adenocarcinoma (PDAC) in terms of expression, influence on cancer cell behavior, and pain correlation.

Summary background data: PDAC is characterized by prominent local nerve alterations and perineural invasion, which frequently affects the extrapancreatic nerve plexus, causing severe pain and precluding curative resection. Artemin, a neurotrophic protein controlling growth, regeneration, and survival of neurons was analyzed to highlight the neuro-cancer interactions in PDAC.

Methods: Artemin and its receptors (GFRalpha3/RET) were studied in PDAC tissues and normal pancreas by Western blot analysis and immunohistochemistry. RNA expression was analyzed in pancreatic tissues (normal, cancer) and pancreatic cancer cell lines by QRT-PCR. To evaluate whether Artemin influences cancer cell proliferation and invasion, MTT-growth and Matrigel-invasion assays were used. In addition, the tissue expression of Artemin was correlated with pain in PDAC.

Results: Artemin and GFRalpha3/RET were both detected at enhanced levels in PDAC compared with normal pancreas, localizing predominantly in hypertrophic nerves and arterial walls, as well as in cancer cells of primary and metastatic lesions. The levels of Artemin and GFRalpha3 did not correlate with pain in PDAC patients. However, Artemin promoted pancreatic cancer cell invasion up to 5-fold, without affecting cancer cell proliferation.

Conclusion: Artemin expression was not associated with pain in PDAC. However by increasing cancer cell invasion, Artemin seems to influence neural invasion and thereby contribute to cancer cell spreading along pancreatic nerves.

FIGURE 1. Artemin and its receptor complex (GFRα3/RET) in pancreatic tissues. A, Artemin-mRNA expression, analyzed by QRT-PCR in pancreatic tissues of healthy organ donors (n = 19) and in pancreatic cancer patients (n = 70), did not show any significant difference. B, Western blot analysis of Artemin, GFRα3, and RET in normal pancreas and in pancreatic cancer showed a 30-fold increase of Artemin, a 20-fold increase of GFRα3, and a 3-fold increase of RET in PDAC. The comparison was made by the ImageJ software. C, Artemin-mRNA expression analyzed by QRT-PCR in the pancreatic cancer cell lines Aspc1, BxPc3, Capan1, Colo-357, MiaPaCa2, Panc1, SU86.86, and T3M4. B, Western blot analysis of Artemin and GFRα3 demonstrating the ubiquitous protein expression in the pancreatic cell lines Aspc1, BxPc3, Capan1, Colo-357, MiaPaCa2, Panc1, SU86.86, and T3M4.

FIGURE 2. Immunoreactivity of Artemin in normal pancreas, pancreatic cancer, and liver metastasis. A and B, Immunostaining of Artemin in normal pancreatic tissue samples was only faintly present in the smooth muscle cells of arteries. In pancreatic cancer (C and D), the immunoreactivity pattern changed dramatically and demonstrated a high accumulation in arteries, nerves, PanIN-lesions, and cancer cells. The same pattern was observed in liver metastasis (E–F) (all images, original magnification ×200).

FIGURE 3. Effect of Artemin on pancreatic cancer cell proliferation. The pancreatic cancer cell lines, T3M4 A, Colo-357 B, MiaPaCa2 C, and SU86.86 D, were exposed to 10 ng/mL and 100 ng/mL Artemin for 24, 48, and 72 hours, respectively. Artemin did not show any significant changes in the proliferation rate of the pancreatic cancer cell lines.

FIGURE 4. Influence of Artemin on invasion and chemoattraction in pancreatic cancer. A, Artemin changed the invasive behavior of the pancreatic cancer cell lines T3M4, Colo-357, MiaPaCa2, and SU86.86 and increased the invasion ratio of all cells in a dose-dependent manner (P < 0.02). B, To analyze the chemotactic potential, Artemin was added in the lower chamber of the Matrigel invasion plate. Hereby, Artemin increased the invasion potential of the pancreatic cancer cells T3M4 and SU86.86 significantly (P < 0.02).

FIGURE 5. Correlation of Artemin and GFRα3 with pain in pancreatic cancer. A, Artemin-mRNA expression analyzed by QRT-PCR in pancreatic tissues of patients suffering from moderate to severe pain (n = 30/Pain III) and of patients without any pain (n = 31/Pain I) revealed no statistically significant differences. B, Western blot analysis of Artemin and GFRα3 from patients in the Pain I group with no pain (n = 8) comparing the Pain III group with moderate to severe pain (n = 8) showed no significant changes.