Abstract 1484: Effects of UCN2 and its receptor CRHR2 in colorectal cancer growth and metastasis.

Background and aims: Colorectal cancer (CRC) represents the third most common form of cancer worldwide and is associated with chronic colonic inflammation. Inflammation is partially regulated by the Corticotropin Releasing Hormone (CRH) family of neuropeptides and their receptors CRHR1 and CRHR2 which are expressed in CNS and periphery including the gastrointestinal tract. However, the effect of CRH system on tumorigenesis is poorly studied and controversial. Up-to-date there are no studies implicating the CRH family of peptides in CRC development and progression. Here, we aim to identify the role the CRH family members play in CRC growth and metastasis in vitro and in vivo and explore the underlying molecular mechanism. Methods: mRNA levels of CRH family members were monitored by qRT-PCR in 46 and 56 normal human colon and CCA biopsies, respectively, as well as in 7 CCA cell lines. CCA cell lines were lentivirally transduced for CRHR2 expression (CRHR2+). The parental and CRHR2+ cells were tested for expression of UCN2, IL-6 and IL-6R using qRT-PCR. Tumor progression was assessed in vitro by proliferation, invasion and migration assays after treatment with IL-6, astressin 2B or Ucn2. Single cell colony formation was assessed by soft agar assay. Expression of pSTAT3 and EMT markers was determined by western blot analysis. An orthotopic transplantation mouse model was used for validation of metastasis in vivo. Results: CCA biopsies and cell lines had almost lost expression of CRHR2 and elevated levels of its selective agonist UCN2, compared to their normal counterparts. CRHR2+ clones had reduced endogenous UCN2 and pro-inflammatory cytokine mRNA levels including IL-6 and its receptor IL-6R. These effects were more pronounced in highly metastatic CRC cell lines and after exogenous administration of UCN2. CRHR2+ clones with downregulated IL-6R levels showed a significantly reduced pSTAT3 levels compared to parental cells, followed by decreased cell proliferation, colony formation, invasion and migration. The above events were able to be reversed by the selective CRHR2 antagonist, astressin 2B, indicating that they were CRHR2 specific. EMT markers were also significantly dysregulated in CRHR2+ clones, suggesting EMT as a downstream target of CRHR2 signaling in CRC. The decreased metastatic potential of CRHR2+ clones, observed in vitro is currently validated in vivo. Conclusions: This is the first demonstration of the protective role of CRHR2 in preventing CRC growth rates and aggressiveness and a novel molecular evidence linking chronic inflammation and CRC through modulation of the Stat3 signaling pathway. Furthermore, monitoring CRHR2 and Ucn2 levels in CRC tissues may benefit early diagnosis of metastasis, and might serve as a novel drug target for prevention and treatment of advanced stages of CRC. Citation Format: Stavroula Baritaki, Jorge Rodriguez, Dimitrios Iliopoulos, Charalabos Pothoulakis. Effects of UCN2 and its receptor CRHR2 in colorectal cancer growth and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1484. doi:10.1158/1538-7445.AM2013-1484