Pancreas carcinoma is one of the most malignant diseases, associated with late and difficult diagnosis and really short survival after diagnosis. Although in most countries effective radiological and other examination methods can be reached, the early diagnosis of pancreas cancer is difficult (1–4). Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated as a hypothalamic neuropeptide acting on the pituitary cAMP release (5,6). The peptide is composed of 38 amino acid residues (PACAP38) and has a shorter form, with only 27 amino acids (PACAP27) (7). Subsequent studies have shown that PACAP is distributed in the entire body, with highest concentrations in the central nervous system and endocrine glands, but it is also present in the cardiovascular, urogenital and gastrointestinal systems (8–13). PACAP has a diverse array of functions via specific PAC1 receptor and VPAC1 and 2 receptors shared with vasoactive intestinal peptide, as well as non-receptorial mechanisms (13,14).

PACAP and its receptors have also been shown in several exocrine glands. The lacrimal gland is innervated by a rich PACAP-ergic fiber plexus (15) and PAC1 receptors are responsible for the activation of tear secretion (16,17). Mammary and salivary glands are also innervated by PACAP-ergic nerves (18–20). In the salivary glands, PACAP induces secretion (21), and enhances protein production while inhibits Ca²⁺ channels (22–24). The exocrine pancreas is histologically similar to serous salivary glands, and the presence of PACAP has also been shown in the exocrine pancreas, where it stimulates acinar lipase secretion (25). Endocrine pancreas, composed of the islets of Langerhans, expresses very high levels of the peptide, similarly to other endocrine glands. Intrainsular PACAP plays a regulatory role in insulin and glucagon secretion and is implied in glucose homeostasis. Pancreatic PACAP has also been implicated in the regulation of beta cell proliferation (26).

Under pathological conditions, a few studies have dealt with changes in PACAP and receptor expression. Previous studies showed that pancreatic over-expression of PACAP increases in cerulein-induced inflammation leading to acute pancreatitis in a mouse model (27). PACAP, along with its receptors, has been shown to be involved in cell proliferation and differentiation both under normal circumstances and in tumourous transformation (28–33). For some tumour cells, PACAP acts as a growth factor (30), while it inhibits growth of others (34). Whether it stimulates growth of pancreatic tumour cells, it is not known at present, however, a PACAP-response gene associated with proliferation and stress response has been described in pancreatic carcinoma (35). Stimulative role of tumour genesis of PACAP is proven by stimulation of c-Fos as well as c-Jun transcription, and PACAP strongly induces proliferation of the rat pancreatic carcinoma cell line AR4-2J via interaction with the G-protein coupled type 1 PACAP/VIP (PV1) receptor (36). PACAP and PAC1 receptor display specific alterations in several different tumour types, such as thyroid papillary carcinoma and testicular cancer (37,38). It is not known how expression of the peptide and its specific receptor changes in pancreatic cancer. Therefore, the aim of the present study was to investigate whether there is a change in the expression of PACAP and its PAC1 receptor in pancreas adenocarcinoma.

In the present study we analysed normal and tumourous pancreas tissues within the same samples for PACAP and PAC1 receptor immunostaining. We observed a diminished expression for both the peptide and its specific receptor in the adenocarcinoma compared to the normal tissue, independent from tumour grade.

Several growth factors play an important role in pancreatic organogenesis and are also involved later in tumourgenesis. Among others, fibroblast growth factor (FGF) has been shown to be involved in the regulation of tumourous cell growth and differentiation in the pancreas (39). FGF receptor IIIb and IIIc play critical roles in the epithelio-mesenchymal transition in spite of no expression in the ductal cells but showing very high expression levels in the islets (40,41). It has been demonstrated that increased nerve growth factor (NGF) expression correlates with poorer prognosis, increased inflammation and pain (42). The involvement of transforming growth factor beta (TGF beta) is unquestionable in tumour growth, including that of the pancreas (43). Overexpression of epidermal growth factor (EGF) occurs in the majority of ductal adenocarcinomas of the pancreas and is associated with poorer prognosis (44–46). The increased insulin-like growth factor expression has been found to be correlated with increased risk of pancreatic cancer (47). There is a continuous, urgent need for novel diagnostic markers for pancreatic cancer (48). The currently available markers have low sensitivity and specificity. Personalized treatment approaches call for more prognostic and treatment-predictive biomarkers (48–50).

PACAP, as a growth factor, plays an important role in the development of the nervous system and several peripheral organs (51–53). It is not surprising, therefore, that certain tumour types also express alterations in PACAP and/or receptor expression. Certain tumours show overexpression of the PACAP-ergic system, while others lack PACAP signalling. In vitro studies have demonstrated that PACAP is able to stimulate or inhibit tumour growth, depending on various factors, such as tumour type, differentiation stage, origin or environmental circumstances (54). For example, PACAP inhibits cell survival in retinoblastoma cells (34), reduces invasiveness in glioblastoma cells (55) and inhibits tumour growth in cervical carcinoma (56). On the other hand, it stimulates cell proliferation in an osteosarcoma cell line (57) and increases the number of viable cells in a colon tumour cell line (58). Even within the same cell line, different effects can be observed depending on exposure time, concentration and other circumstances. This dual effect has been described in a prostate cancer cell line, where short exposure to PACAP induces cell proliferation, while long-term exposure induces proliferation arrest (59). In a human retinoblastoma cell line, nanomolar concentrations of PACAP do not affect cell viability, while higher concentrations decrease cell survival (34).

PACAP/VIP receptors are known to play a leading role in cancer genesis and the VIP/PACAP receptors are expressed in the most frequently occurring human tumours (breast, prostate, ductal carcinoma of the pancreas, lung, colon, stomach, liver, and urinary bladder, lymphomas and meningioma). In these cases the receptors are predominantly VPAC1 type. On the other hand leiomyomas predominantly express VPAC2 receptors, whereas paraganglioma, pheochromocytoma, and endometrial carcinomas preferentially express PAC1 receptors (60). Recent studies have shown that VIP/PACAP-receptor expression can be found in only 65% of pancreatic ductal carcinomas (30). Both VPAC1 and 2 receptors have been identified in pancreatic tumour samples (30). Overexpression of these receptors (61) explains the attempts for the clinical use of radiolabelled VIP-analogues in various cancer types, including pancreas adenocarcinoma (30,62,63). However, contradictory data have also been published, as according to the observations of Hessenius and coworkers (62), no imaging was seen with radiolabeled-VIP-analogues in pancreatic cancer patients, and in vitro binding studies in these tumours did not confirm overexpression of VPAC1. We found PAC1 receptor expression in the exocrine pancreas in nearly all cases, but very weak expression in the tumourous parts. Changes in PACAP expression have been shown in a few tumours by radioimmunoassay and immunohistochemistry (64). In earlier studies, we described lower PACAP tissue levels in lung, kidney and colon cancer, but higher levels in prostate cancer (64,65). A changed staining pattern has been described in different human testicular cancers (38) and in human thyroid papillary carcinoma (37). In the present study we observed that PACAP expression was weak in normal tissues in the exocrine pancreas, and nearly absent in the adenocarcinoma parts of the tissue samples. The limitation of our study is that we cannot draw final conclusion at the moment whether the reduction of PACAP and PAC1 receptor expression is a consequence of the adenocarcinoma development or the reduced PACAP signaling plays a role in pancreatic carcinogenesis. This should be further explored in future studies.

In summary, we found that both PACAP and PAC1 receptor expression is markedly decreased in human pancreatic ductal adenocarcinoma tissue samples, while staining remained strong in the endocrine islets. This suggests that decrease or lack of the PAC1 receptor/PACAP signalling may contribute to tumour growth and/or differentiation, details of which must be further explored.