Novel effective treatment is direly needed for patients with pancreatic ductal adenocarcinoma (PDAC). Therapeutics that target the driver mutations, especially the KRAS oncoprotein and its effector cascades, have been ineffective. It is increasing clear that the extensive fibro-inflammatory stroma (or desmoplasia) of PDAC plays an active role in the progression and therapeutic resistance of PDAC. The desmoplastic stroma is composed of dense extracellular matrix (ECM) deposited mainly by the cancer-associated-fibroblasts (CAFs) and infiltrated with various types of immune cells. The dense ECM functions as a physical barrier that limits tumor vasculatures and distribution of therapeutics to PDAC cells. In addition, mounting evidence have demonstrated that both CAFs and ECM promote PDAC cells aggressiveness through multiple mechanisms, particularly engagement of the epithelial-mesenchymal transition (EMT) program. Acquisition of a mesenchymal-like phenotype renders PDAC cells more invasive and resistant to therapy-induced apoptosis. Here, we critically review seminal and recent articles on the signaling mechanisms by which each stromal element promotes EMT in PDAC. We discussed the experimental models that are currently employed and best suited to study EMT in PDAC, which are instrumental in increasing the chance of successful clinical translation.

Epithelial-to-mesenchymal transition (EMT) is linked to tumor invasion, drug resistance and aggressive disease and this is largely dependent on the cell's microenvironment. Acriflavine (ACF) is an old antibacterial drug recently also suggested as anticancer agent and HIF inhibitor. We wanted to study the effect of acriflavine on EMT in different human cancer models. Pancreatic cancer cells (Panc-1) were exposed to TGF-β1 or cobalt chloride (to mimick severe hypoxia) to induce EMT. For our third model we exposed HepG2 liver cancer cells to sorafenib which resulted in development of acquired drug resistance with strong features of EMT and aggressive behavior. These models were morphologically and functionally (invasion assay) characterized. Markers of EMT were determined using qRT-PCR and Western blotting. Transcriptome analysis was performed following gene expression determination and combining the iRegulon tool and Gene Set Enrichment Analysis (GSEA). We made the following observations: (1) acriflavine inhibited EMT based on changes in cell morphology, invasive capacities and markers of EMT (at protein and gene expression level). (2) Transcriptome analysis revealed potent inhibition of ATF4 target genes and of the unfolded protein response. We showed that acriflavine blocked eIF2a phosphorylation and reduced ATF4 translation thereby inhibiting the PERK/eIF2a/ATF4 UPR pathway. (3) ACF restored drug sensitivity of cells that obtained acquired resistance. Conclusions: We identified acriflavine as a potent inhibitor of EMT and the UPR, thereby re-sensitizing the cancer cells to antineoplastic drugs.

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Purpose: All currently available therapies for the treatment of pancreatic ductal adenocarcinoma (PDAC) show limited success. PDACs fast progression depends on the tumor characteristics and can be influenced by the microenvironment. The antibacterial drug acriflavine (ACF) has been shown to have anti-cancer activities in cell lines. Materials and Methods: To understand the working mechanism of ACF on cancer progression and tumor-stromal interplay, we evaluated pancreatic cancer in cell culture (Panc-1) (morphology, cell invasion and RNA expression) and the macrophage cell line THP1 (representing innate immune stromal cells). In the translational arm, the activity of ACF on xenograft models of human PDAC tumors representing different clinical subclasses was investigated (tumor growth, morphology and immunofluorescence, RNA expression and pathway analysis). Results: In vitro, ACF reduces epithelial-to-mesenchymal transition (EMT) and invasion of Panc-1 cells and shifts macrophage polarization to a M1-like anti-tumoral phenotype. At non-toxic concentrations, ACF downregulates cell metabolism. In xenografts, effect on tumor growth and metabolism could be confirmed; however, the innate immune stromal cells did not respond. Importantly, only in the moderately differentiated PDAC model, ACF could significantly suppress tumor growth and not in the fast-growing EMT-high model. Pathway analysis shows that ACF highly significantly downregulates metabolic pathways, especially OXPHOS and MYC/cell proliferation pathways in xenografts. Conclusion: ACF, with known pleiotropic effects on cancer cells, is in this study shown to be an attractive therapeutic based on its novel observed metabolic activity. Repurposing this compound for cancer treatment should be in the setting with other targeting agents, which offers reduced chance of resistance development in PDAC. Further evaluation should best be done in biological complex models such as human xenografts or syngeneic cancer models.