Federico Bussolino, M.D. Ph.D.
Full Professor of Biochemistry University of Torino, School of Medicine
Identification of early biomarkers of pancreatic ductal carcinoma (PDAC).
Elegant tracking studies of the genetic evolution of PDACs indicate at least 15 years between the occurrence of the initiating mutation and the acquisition of metastatic ability. This result demonstrates a broad time window of opportunity for early detection to prevent deaths from metastatic disease. We reasoned that an early biomarker had to play a necessary role in the onset of the disease and it had to be preferentially released from the cells. Therefore we performed a whole transcriptomic analysis of the early in situ carcinomas (PanIN) isolated by laser capture microdissection from a genetically engineered mouse model characterized by the expression of KRASG12V in acinar cells through the use of the elastase promoter (Elas-K-RasG12V).
RNAs highly expressed in PanIN encode proteins involved in axon guidance cues and synaptogenesis that have been recently demonstrated to be altered in PDAC. We focused on neuroligins (Nlg), a family of adhesive molecules regulating synaptic activity. Anti-pan-Nlgs antibody showed a faint membrane signal of acinar and ductal cells whereas PanIN cells displayed a stronger staining that dropped in PDAC. Similar results were obtained in human lesions. By analysing the expression of the 5 Nlgs we visualized a minute population of acinar cells carrying KRasG12V and expressing Nlg-2 associated with stemness markers (DCLK1, CD24, CD44, and CXCR4). The current hypothesis of the pathogenesis of PDAC relies on the differentiation of acinar precursors carrying KRAS mutation into ductal cells (acinar-ductal metaplasia). We demonstrated that KRasG12V acinar cells isolated from Elas-KRasG12V underwent in vitro acinar ductal metaplasia with an increased expression of Nlg-2. An isogenic cell line carrying KRasG12V overexpressed Nlg-2 if compared with wild-type cells. Lossof-function experiments showed that Nlg-2 ablation halted acinar-ductal metaplasia, supporting the idea that Nlg-2 is necessary in the early phase of PDAC. Then, we considered the possibility that Nlg-2 was shed by the cells, supported by the presence of a Disintegrin And Metalloproteinase (ADAM) proteolytic cleavage site in the extracellular domain. Nlg-2 accumulated in the serum-free supernatant of pancreatic PT45 human cell line overexpressing Nlg-2. This phenomenon is blocked by ADAM inhibitors TAPE-2 and GM 6001.
Conclusions and perspectives:
We reported that Nlg2 (i) has an early role in PDAC by promoting the acinar-ductal metaplasia of precursors; (ii) is shed by an ADAM-dependent mechanism. To further support the role of Nlg2 as an early biomarker in PDAC, we will continue the following activities: (i) demonstration of the role of Nlg-2 in stemness features of acinar precursors undergoing acinar-ductal metaplasia; (ii) analysis of PDAC progression in Elas-K-RasG12V model crossed-back with Nlg-2-/- mice (iii) generation of mAb anti-Nlg2 to set-up an ELISA for Nlg-2 quantification in biological fluids; (iv) increase of human samples to analyse Nlg-2 expression and activities.