Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer

Background: There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding-and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. Methods: mRNA in situ hybridisation and immunostaining for E-cadherin, beta-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. Results: Tumour-budding cells showed increased levels of ZEB1 (Po0.0001) and ZEB2 (P = 0.0119) and reduced E-cadherin and b-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous beta-catenin in the main tumour (P = 0.0009) and tumour buds (P = 0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P = 0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P = 0.0084; 0.0250 and 0.0029, respectively) and high PTB (P = 0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P = 0.03), lymphatic invasion (P = 0.0172) and lymph node metastasis (P = 0.0152). Conclusions: In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.