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Epigenetic reprogramming using 5-azacytidine promotes an anti-cancer response in pancreatic adenocarcinoma cells.

ABSTRACT: Curative management of pancreatic adenocarcinoma is limited because this malignancy remains resistant to most chemotherapeutic drugs. Strategies that reverse epigenetic alterations offer a unique opportunity for cancer cell reprogramming, which is valuable for development of new treatments. The aim of this work was to reprogram pancreatic ductal adenocarcinoma (PDAC) cells toward a less aggressive and drug-responsive phenotype. The process applied is called "epigenetic reprogramming". To evaluate the efficiency of PDAC epigenetic reprogramming, we assessed tumor growth and drug response in PANC-1 cells after exposure to non-cytotoxic doses of the demethylating agent 5-azacytidine (5-AZA). Here, we showed that an epigenetic regimen using 5-AZA promoted an anti-cancer response by inhibiting PDAC tumor growth in vivo after the engraftment of treated cells. Remarkably, the subsequent addition of gemcitabine (GEM) to the 5-AZA-mediated reprogramming resulted in a marked growth inhibition effect in GEM-resistant pancreatic cancer cells. We observed that various characteristic peptides expressed in the pancreas, which included the antiproliferative hormone somatostatin (SST) and the SST receptor 2 (SSTR2), were significantly upregulated in the epigenetically reprogrammed PDAC cells. The inhibitory effect of octreotide (OCT), an SST analog, was tested on PDAC cells and found to be improved after cell reprogramming. Furthermore, we found that SST gene expression restoration following 5-AZA treatment or following knockdown of the DNA methyltransferase (DNMT) 1 enzyme was associated with the reversion of SST epigenetic silencing through regional CpG demethylation. Lastly, we confirmed the efficacy of 5-AZA-based epigenetic reprogramming in vivo using a PDAC tumor growth model. In conclusion, this study demonstrates that epigenetic reprogramming using the demethylating compound 5-AZA shows anti-cancer effects in PANC-1 cells and is potentially attractive for the treatment of solid tumors.

SUBMITTER: Gailhouste L

PROVIDER: S-EPMC5920091 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPancreatic ductal adenocarcinoma (PDAC) tends to undergo distant metastasis, especially liver metastasis, leading to a poor prognosis. Metabolic remodelling and epigenetic reprogramming are two important hallmarks of malignant tumours and participate in regulating PDAC tumorigenesis and metastasis. However, the interaction between these two processes during PDAC metastasis has not been fully elucidated.MethodsWe performed metabolomics analysis to identify the critical metabolites associated with PDAC liver metastasis and focused on guanidinoacetic acid (GAA). Intracellular GAA content was significantly increased in liver metastatic PDAC cells compared to primary cancer cells in mouse xenograft tumour models. The effects of GAA supplementation and glycine amidinotransferase (GATM) knockdown on PDAC metastasis were assessed by analysing cell migration, filopodia formation, epithelial-mesenchymal transition (EMT), and in vivo metastasis in different cell and animal models. Next, ChIP‒qPCR, 3C‒qPCR, and CRISPRi/dCas9-KRAB experiments were used to validate the "epigenome-metabolome" mechanism. Finally, the results of in vitro approaches, including RNA-seq, CUT&RUN, RT‒qPCR, and western blot analyses, as well as luciferase reporter gene assay and transwell assay, revealed the GAA-c-Myc-HMGA axis and transcription-activating histone modifications reprogramming.ResultsA high level of intracellular GAA was associated with PDAC liver metastasis. GAA could promote the migration, EMT, and liver metastasis of pancreatic cancer cells in vitro and in vivo. Next, we explored the role of GATM-mediated de novo GAA synthesis in pancreatic cancer metastasis. High expression of GATM was positively correlated with advanced N stage in PDAC. Knockdown of GATM significantly reduced the intracellular level of GAA, suppressed EMT, and inhibited PDAC liver metastasis, and these effects were attenuated by GAA supplementation. Mechanistically, we identified the active enhancers looped to the Gatm gene locus that promoted GATM expression and PDAC liver metastasis. Furthermore, we found that GAA promoted cell migration and EMT by regulating c-Myc-mediated high mobility group AT-hook protein expression. Moreover, GAA increased the H3K4me3 modification level by upregulating histone methyltransferases, which induced the transcription of metastasis-related genes, including Myc.ConclusionsThese findings revealed the critical role of the epigenome-metabolome interaction in regulating PDAC liver metastasis and suggested potential therapeutic strategies targeting GAA metabolism and epigenetic regulatory mechanisms.

Project description:Chronic hepatitis B virus (HBV) infections remain a health burden affecting ~250 million people worldwide. Thus far, available interferon-alpha (IFNα)-based therapies have shown unsatisfactory cure rates, and alternative therapeutic molecules are still required. However, their development has been hampered because accessible cell models supporting relevant HBV replication and appropriate antiviral activity are lacking. Strategies that reverse epigenetic alterations offer a unique opportunity for cell reprogramming, which is valuable for restoring altered cellular functions in human cell lines. This work aimed to investigate the feasibility of converting HepG2 cells that stably overexpress the HBV entry receptor (sodium/taurocholate cotransporting polypeptide, NTCP) toward IFNα-responsive cells using epigenetic reprogramming. Herein, we showed that an epigenetic regimen with non-cytotoxic doses of the demethylating compound 5-azacytidine restored the anti-HBV action of IFNα in epigenetically reprogrammed HepG2-NTCP-C4 cells, named REP-HepG2-NTCP cells. Thus, a significant inhibition in HBV DNA levels was measured in REP-HepG2-NTCP cells after IFNα treatment. This inhibitory effect was associated with the enhancement of IFNα-mediated induction of critical interferon-stimulated genes (ISGs), which was limited in non-reprogrammed cells. In particular, our data indicated that re-expression of 2'-5'-oligoadenylate synthetase 1 (OAS1) and interferon regulatory factor 9 (IRF9) was the result of an epigenetically driven unmasking of these genes in reprogrammed cells. At last, we evaluated the therapeutic potential of the IFN analog CDM-3008 in REP-HepG2-NTCP cells and demonstrated the efficiency of this chemical compound in triggering ISG induction and HBV inhibition. In summary, this study shows that epigenetic reprogramming promotes the IFNα response in HBV-infected cells and is potentially attractive for cell-based experimental screening of IFN-like compounds.

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Epigenetic reprogramming using 5-azacytidine promotes an anti-cancer response in pancreatic adenocarcinoma cells.

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