br Methods br Results br Discussion With this study
Discussion With this study, we provided evidence that acriflavine can interfere with EMT and UPR, two well-described pathways associated with cancer aggressiveness. And, because drug resistance is also clearly associated with the mesenchymal state , blocking of the epithelial-to-mesenchymal transition is an attractive goal in ACF cancer therapy. We could show that acriflavine, an old antibiotic recently identified as anticancer agent and HIF inhibitor , efficiently inhibits EMT in vitro. Expression of several important epithelial and mesenchymal markers that were altered by EMT induction can be restored using acriflavine (gene and protein level). At the morphological level, we showed that induced Panc-1 Bioactive Compound Library (CoCl2 or TGF-β1: mesenchymal) following acriflavine treatment regain closer cell–cell contact as is seen in the non-stimulated Panc-1 cells (epithelial). In HepG2S1 cells, the phenotype is much more pronounced and “fixed” probably due to numerous genetic and epigenetic alterations present in these cells, which was already shown in similar models of mesenchymal differentiation upon acquired drug resistance . Nevertheless, the invasive capacities of stimulated Panc-1 cells and of HepG2S1 could be abrogated by a noncytotoxic concentration of acriflavine. Much more than the expression of a single epithelial or mesenchymal marker, this effect on the cellular behavior is crucial to demonstrate EMT inhibitory activity. Another remarkable effect of acriflavine is the inhibition of the unfolded protein response. The role of the UPR is to limit cellular damage during stress, explaining why the UPR has been linked to cancer drug resistance . However, this mechanism equally drives the cell to apoptosis when the stress is too severe . Given this dual role, the question rises whether either inhibition or stimulation of the UPR for cancer therapy is most desirable. With this study, we showed that acriflavine causes dephosphorylation of eIF2a, hereby inhibiting the ATF4 transcriptional program. ATF4 is one of the central transcription factors in the UPR induced by severe hypoxia or anoxia, independent of HIF signaling . ATF4 is generally regarded as a pro-survival factor associated with resistance to anti-cancer drugs, which makes it an attractive target . Several approaches to target ATF4 have been suggested, including the inhibition of upstream factors PERK and eIF2a . To the best of our knowledge, no drugs have yet demonstrated to clearly abrogate the ATF4 transcriptional program. Moreover, several genes regulated by ATF4 and affected by ACF, such as carbonic anhydrase 9, are attractive targets for cancer therapy . Acriflavine can thus be considered as a broad hypoxia pathway inhibitor in in vitro cancer models. Earlier, the drug was identified as a HIF inhibitor by preventing dimerization of the two HIF-1 subunits . We now show that ACF also blocks the alternative hypoxia pathway via ER stress causing abrogation of hypoxic cell survival . To be noted, ACF suppresses predominantly the expression of cytoplasmic aminoacyl t-RNA synthetases and not the mitochondrial forms (see Supplementary Table 4) suggesting that the mitochondria and energy supply of the cell are not affected. Furthermore, in two models, hepatoma cells resistant to sorafenib and pancreatic cancer cells resistant to gemcitabine, we could demonstrate a shift of drug sensitivity towards the parental cell line. Together, these action mechanisms of ACF result in diminished invasion and resensitization of cancer cells in vitro. Being an in vitro study it has some limitations before it can be translated to the clinic. We investigated several cell models in which the results on EMT or ACF are not always identical. We selected the models to be complementary, we saw that EMT induction by stimulation (TGF-β1 or CoCl2) is functional not very different from the drug resistance models although certain molecular features are not exactly the same. The same variation is to be expected when in vivo different microenvironmental factors induce a cancer cells to EMT. We then looked for features that could be repeated in the different models of aggressive cancer. Our conclusions are therefore based on the linkage of transcriptome data with alterations in cellular programs EMT and UPR. Although these findings are very suggestive for a causal relationship, additional mechanistic studies are needed to definitely confirm our hypotheses. However, recent studies have shown that PERK/eIF2a activation is necessary for EMT cells to invade and metastasize providing a link between the two concepts . Moreover, ATF4 knock-down impedes hypoxia induced EMT in gastric cancer cell lines .