Epigenetic loss of SLIT2 leads to an autocrine-to-paracrine switch of the SLIT2/ROBO1 signaling axis in pancreatic cancer

Abstract

Guidance molecules from the Netrin, Slit, Ephrin, and Semaphorin gene families were originally described as cues for the directional guidance of axons in the developing nervous system. More recently, members of these families have been found to have critical roles in epithelial development, angiogenesis and cancer. The SLIT2/ROBO1 signaling axis has properties of a potential tumor suppressor pathway via the inhibition of epithelial cell growth, directional migration, ductal morphogenesis, and is epigenetically silenced in lung, colon and breast cancers. We proposed that changes in SLIT2 and ROBO1 expression in pancreatic ductal adenocarcinoma may mediate ductal expansion following the conversion of PanIN precursor lesions to invasive carcinoma. The SLIT2 receptor, ROBO1, is a member of the immunoglobulin (Ig) superfamily and is required for lung and mammary development in mammals. ROBO1 has an alternative splice variant, DUTT1, and these two variants have different initial exons and initiating codons which may suggest the two proteins have distinct functions. In our studies, we found that all pancreatic cancer cell lines and primary pancreatic cancer specimens express only the DUTT1 isoform. We also determined that as pancreatic cancer cell lines become KRAS-independent, ROBO1 expression increases. Furthermore, using immunohistochemistry (IHC), we found that ROBO1 protein expression in primary pancreatic cancer tissue specimens is localized to the ductal compartment with no stromal staining seen. In normal pancreas, ROBO1 expression is weak while its ligand SLIT2 is strongly expressed in both the acinar and ductal compartments in vitro and in vivo. Mammals encode three SLIT genes (SLIT1-3). The secreted SLIT2 protein is not diffusible, but has a cleavage site within its EGF-like repeats creating two fragments which allow it to act either as a short or long range guidance cue with each fragment appearing to have its own cell-association characteristics. The 5’ promoter of SLIT2 has been shown to be methylated resulting in gene silencing in early stages of several epithelial cancers suggesting a possible tumor suppressor role. miR-218-1 is an intronic microRNA found between exons 15 and 16 of the SLIT2 gene and targets a complimentary sequence in the ROBO1 3’ untranslated region (UTR) indicative of a potential regulation of receptor availability in the presence of ligand. In our studies, we determined that the KRAS-dependent pancreatic cancer cell lines express SLIT2 and ROBO1 in a cell autonomous manner. The KRAS-independent cell lines, however, have silenced SLIT2 and miR-218-1 expression. Using IHC we found that high levels of SLIT2 are seen in normal pancreas localized to the acinar and ductal compartments. Reduced SLIT2 expression is seen in primary pancreatic cancer tissue specimens. We confirmed that loss of SLIT2 mRNA in KRAS-independent lines was due to DNA hypermethylation shown by methylation specific PCR and Sequenom analysis. Chromatin immunoprecipitation analysis shows that silencing histone marks are found in the 5’ promoter of the SLIT2 gene in KRAS-independent lines. Treatment with demethylating agents reactivate SLIT2 and miR-218-1 expression suggesting that epigenetic mechanisms controlling the SLIT2 promoter also regulate miR-218-1 expression. Overall, our data establishes that the SLIT2/ROBO1 signaling axis is a dynamic pathway in pancreatic cancer that can act in the tumor expansion and progression along intrapancreatic neurons that express the SLIT2 ligand.