Abstract
Neurotrophic factors possess an emerging role in the pathophysiology of several gastrointestinal disorders, regulating innervation, pain sensation and disease-associated neuroplasticity. Here, we aimed at characterizing the role of the neurotrophic factor neurturin (NRTN) and its receptor glial-cell-line-derived neurotrophic factor receptor alpha-2 (GFRα-2) in pancreatic cancer (PCa) and pancreatic neuropathy. For this purpose, NRTN and GFRα-2 were studied in normal human pancreas and PCa tissues via immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, immunoblotting and correlated to abdominal pain. The impact of NRTN/GFRα-2 on PCa cell (PCC) biology was investigated via exposure to hypoxia, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide viability and matrigel invasion assays in native and specific small interfering RNA-silenced PCCs. To assess the influence of NRTN on pancreatic neuroplasticity and neural invasion (NI), its impact was explored via an in vitro 'neuroplasticity assay' and a 3D neural migration assay. NRTN and GFRα-2 demonstrated a site-specific upregulation in PCa, predominantly in nerves, PCCs and extracellular matrix. Patients with severe pain demonstrated higher intraneural GFRα-2 immunoreactivity than patients with no pain. PCa tissue and PCCs contained increased amounts of NRTN, which was suppressed under hypoxia. NRTN promoted PCC invasiveness, and silencing of NRTN limited both PCC proliferation and invasion. Depletion of NRTN from PCa tissue extracts and PCC supernatants decreased axonal sprouting in neuronal cultures but did not influence glial density. Silencing of NRTN in PCCs boosted NI. We conclude that increased NRTN/GFRα-2 in PCa seems to promote an aggressive PCC phenotype and neuroplasticity in PCa. Accelerated NI following NRTN suppression constitutes a novel explanation for the attraction of PCC to nerves in the hypoxic PCa tumor microenvironment. PCa is characterized by intrapancreatic neuroplasticity and NI. Here, we show that PCC produce the neurotrophic factor NRTN, which reinforces their biological properties, triggers neuroplastic alterations, NI and influences pain sensation via the GFRα-2 receptor.
Highlights
The pancreas is a densely innervated organ, which is surrounded byIn all pancreatic ductal adenocarcinoma (PCa) patients, individual pain degree was prospectively registered and calculated prior to the operation, as described previously [20].numerous neural networks and receives input over splanchnic/vagal nerves and from intrapancreatic cholinergic ganglia [1,2]
NRTN and glial-cell-line-derived neurotrophic factor receptor alpha-2 (GFRα-2) are upregulated in PCa We first investigated the distribution of NRTN and GFRα-2 in normal human pancreas (NP) and PCa
The present study was designed to elucidate the role of the NRTN/ GFRα-2 axis in the pathophysiology of PCa and especially in pancreatic neuropathy
Summary
In all PCa patients, individual pain degree (no pain/group 0, mild pain/group I and severe pain/group II) was prospectively registered and calculated prior to the operation, as described previously [20]. Numerous neural networks and receives input over splanchnic/vagal nerves and from intrapancreatic cholinergic ganglia [1,2]. Despite this naturally rich innervation pattern, one can recognize that nerves in pancreatic ductal adenocarcinoma (PCa) undergo a prominent. Alexa® Fluor 488 and 594 antibodies (Invitrogen, Karlsruhe, Germany) in combination with 4′,6-diamidino-2-phenylindole nuclear stain were utilized, as described previously [22]. Histopathological analysis was performed by two independent observers (K.W., I.E.D.) blinded to patient data, followed by resolution of any differences The degree of immunoreactivity on each section and each tissue substructure was scored and added using a numerical scale (0: no staining, 1: weak staining, 2: moderate staining, 3: strong staining) and averaged among all patients to obtain the ‘mean tissue immunoreactivity score’
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