In vitro, in vivo and ex vivo demonstration of the antitumoral role of hypocretin-1/orexin-A and almorexant in pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is still the poorest prognostic tumor of the digestive system. We investigated the antitumoral role of orexin-A and almorexant in PDAC. We analyzed the orexin receptor type 1 (OX1R) expression by immunohistochemistry in human normal pancreas, PDAC and its precursor dysplastic intraepithelial lesions. We used PDAC-derived cell lines and fresh tissue slices to study the apoptotic role of hypocretin-1/orexin-A and almorexant in vitro and ex vivo. We analyzed in vivo the hypocretin-1/orexin-A and almorexant effect on tumor growth in mice xenografted with PDAC cell lines expressing, or not, OX1R. Ninety-six percent of PDAC expressed OX1R, while adjacent normal exocrine pancreas did not. OX1R was expressed in pre-cancerous lesions. In vitro, under hypocretin-1/orexin-A and almorexant, the OX1R-positive AsPC-1 cells underwent apoptosis, abolished by the tyrosine phosphatase SHP2 inhibitor, NSC-87877, whereas the OX1R-negative HPAF-II cell line did not. These effects were mediated by phosphorylation of OX1R and recruitment of SHP2. Ex vivo, caspase-3 positive tumor cells were significantly higher in fresh tumour slices treated 48h with hypocretin-1/orexin-A, as compared to control, whereas cellular proliferation, assessed by Ki-67 index, was not modified. In vivo, when AsPC-1 cells or patient-derived cells were xenografted in nude mice, hypocretin-1/orexin-A or almorexant, administrated both starting the day of cell line inoculation or after tumoral development, strongly slowed tumor growth. Hypocretin-1/orexin-A and almorexant induce, through OX1R, the inhibition of PDAC cellular growth by apoptosis. Hypocretins/orexins and almorexant might be powerful candidates for the treatment of PDAC.

The Anti-tumoral Properties of Orexin/Hypocretin Hypothalamic Neuropeptides: An Unexpected Therapeutic Role

“…It should be noted that OX1R was not expressed in normal pancreas (acini and ducts) except in the Langerhans islets (Figure 3A ) in which orexins could play a role in insulin secretion ( 8 ). This expression in tumoral tissue was independent of patient age, gender, tumor size, and lymph node metastasis ( 46 ). The use of AsPC-1 cell line derived from human PDAC revealed that OxA was able to strongly inhibit cell growth by the SHP2-induced apoptosis ( 46 ).…”

“…This expression in tumoral tissue was independent of patient age, gender, tumor size, and lymph node metastasis ( 46 ). The use of AsPC-1 cell line derived from human PDAC revealed that OxA was able to strongly inhibit cell growth by the SHP2-induced apoptosis ( 46 ). Moreover, the treatment by OxA of tumor slices obtained from patients and maintained in culture, induced the activation of caspases-3 in tumoral tissue demonstrating that OxA was able to induce apoptosis in PDAC ( 46 ).…”

“…(•), control mice injected with PBS; (◦), treated mice injected with OxA. The sources of graphs were based on Voisin et al ( 44 ), Dayot et al ( 46 ), and Chartrel et al ( 47 ).…”

“…Recently, the OX1R expression was detected in 70/73 human PDAC (96 %) and in 83/103 human pancreatic neuroendocrine tumors ( 46 ). It should be noted that OX1R was not expressed in normal pancreas (acini and ducts) except in the Langerhans islets (Figure 3A ) in which orexins could play a role in insulin secretion ( 8 ).…”

“…The use of AsPC-1 cell line derived from human PDAC revealed that OxA was able to strongly inhibit cell growth by the SHP2-induced apoptosis ( 46 ). Moreover, the treatment by OxA of tumor slices obtained from patients and maintained in culture, induced the activation of caspases-3 in tumoral tissue demonstrating that OxA was able to induce apoptosis in PDAC ( 46 ). In preclinical model consisting in sub-cutaneous xenografted AsPC-1 cells in nude mice, OxA reduced significantly the tumor growth (Figure 2B ).…”