TAp73 Inhibits EMT and Cell Migration in Pancreatic Cancer Cells through Promoting SMAD4 Expression and SMAD4-Dependent Inhibition of ERK Activation

SIMPLE SUMMARY: Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease due to early metastatic spread, late diagnosis and the lack of efficient therapies. The aim of our study was to reveal if transcriptionally active p73 (TAp73), a homolog of the well-known tumor suppressor p53, inhibits tumor progression through promoting canonical TGF-β/Smad signaling and by preventing non-canonical extracellular signal-regulated kinases (ERK)1/2-mediated TGF-β signaling. Using PDAC-derived tumor cell lines, we showed that TAp73 suppresses epithelial-mesenchymal transition by inducing the expression of epithelial markers while suppressing that of mesenchymal markers. We further demonstrated that TAp73 upregulates the expression of the TGF-β signaling intermediate SMAD4 and that SMAD4 acts a mediator of TAp73-induced inhibition of ERK activation and cell motility. Measuring the levels of TAp73 and/or SMAD4 could help to predict whether TGF-β preferentially uses an oncogenic or a tumor suppressive pathway in a given patient and at a specific time. ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease due to early metastatic spread, late diagnosis and the lack of efficient therapies. A major driver of cancer progression and hurdle to successful treatment is transforming growth factor (TGF)-β. Recent data from pancreatic cancer mouse models showed that transcriptionally active p73 (TAp73), a p53 family member, inhibits tumor progression through promoting tumor suppressive canonical TGF-β/Smad signaling, while preventing non-canonical TGF-β signaling through extracellular signal-regulated kinases (ERK)1/2. Here, we studied whether this mechanism also operates in human PDAC. Using the PDAC-derived tumor cell lines PANC-1, HPAFII and L3.6pl, we showed that TAp73 induces the expression of the epithelial marker and invasion suppressor E-cadherin and the common-mediator Smad, SMAD4, while at the same time suppressing expression of the EMT master regulator SNAIL and basal and TGF-β1-induced activation of ERK1 and ERK2. Using dominant-negative and RNA interference-based inhibition of SMAD4 function, we went on to show that inhibition of ERK activation by TAp73 is mediated through SMAD4. Intriguingly, both SMAD4 and the α isoform of TAp73—but not the β isoform—interfered with cell migration, as shown by xCELLigence technology. Our findings highlighted the role of TAp73-SMAD4 signaling in tumor suppression of human PDAC and identified direct inhibition of basal and TGF-β-stimulated pro-invasive ERK activation as an underlying mechanism.