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Epigenetic reprogramming of primary pancreatic cancer cells counteracts their in vivo tumourigenicity.

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) arises through accumulation of multiple genetic alterations. However, cancer cells also acquire and depend on cancer-specific epigenetic changes. To conclusively demonstrate the crucial relevance of the epigenetic programme for the tumourigenicity of the cancer cells, we used cellular reprogramming technology to reverse these epigenetic changes. We reprogrammed human PDAC cultures using three different techniques - (1) lentivirally via induction of Yamanaka Factors (OSKM), (2) the pluripotency-associated gene OCT4 and the microRNA mir-302, or (3) using episomal vectors as a safer alternative without genomic integration. We found that induction with episomal vectors was the most efficient method to reprogram primary human PDAC cultures as well as primary human fibroblasts that served as positive controls. Successful reprogramming was evidenced by immunostaining, alkaline phosphatase staining, and real-time PCR. Intriguingly, reprogramming of primary human PDAC cultures drastically reduced their in vivo tumourigenicity, which appeared to be driven by the cells' enhanced differentiation and loss of stemness upon transplantation. Our study demonstrates that reprogrammed primary PDAC cultures are functionally distinct from parental PDAC cells resulting in drastically reduced tumourigenicity in vitro and in vivo. Thus, epigenetic alterations account at least in part for the tumourigenicity and aggressiveness of pancreatic cancer, supporting the notion that epigenetic modulators could be a suitable approach to improve the dismal outcome of patients with pancreatic cancer.

SUBMITTER: Khoshchehreh R

PROVIDER: S-EPMC6756074 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Epigenetic reprogramming of primary pancreatic cancer cells counteracts their in vivo tumourigenicity.

Khoshchehreh Reyhaneh R Totonchi Mehdi M Carlos Ramirez Juan J Torres Raul R Baharvand Hossein H Aicher Alexandra A Ebrahimi Marzieh M Heeschen Christopher C

Oncogene 20190715 34

Pancreatic ductal adenocarcinoma (PDAC) arises through accumulation of multiple genetic alterations. However, cancer cells also acquire and depend on cancer-specific epigenetic changes. To conclusively demonstrate the crucial relevance of the epigenetic programme for the tumourigenicity of the cancer cells, we used cellular reprogramming technology to reverse these epigenetic changes. We reprogrammed human PDAC cultures using three different techniques - (1) lentivirally via induction of Yamanak ...[more]

PMID: 31308488

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Project description: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.

Project description:BackgroundExtracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter- α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive.MethodsITIH5 expression was analyzed using the public TCGA portal. ITIH5-overexpressing single-cell clones were established based on T47D and MDA-MB-231 cell lines. Colony formation, growth, apoptosis, migration, matrix adhesion, traction force analyses and polarization of tumor cells were studied in vitro. Tumor-initiating characteristics were analyzed by generating a metastasis mouse model. To identify ITIH5-affected pathways we utilized genome wide gene expression and DNA methylation profiles. RNA-interference targeting the ITIH5-downstream regulated gene DAPK1 was used to confirm functional involvement.ResultsITIH5 loss was pronounced in breast cancer subtypes with unfavorable prognosis like basal-type tumors. Functionally, cell and colony formation was impaired after ITIH5 re-expression in both cell lines. In a metastasis mouse model, ITIH5 expressing MDA-MB-231 cells almost completely failed to initiate lung metastases. In these metastatic cells ITIH5 modulated cell-matrix adhesion dynamics and altered biomechanical cues. The profile of integrin receptors was shifted towards β1-integrin accompanied by decreased Rac1 and increased RhoA activity in ITIH5-expressing clones while cell polarization and single-cell migration was impaired. Instead ITIH5 expression triggered the formation of epithelial-like cell clusters that underwent an epigenetic reprogramming. 214 promoter regions potentially marked with either H3K4 and /or H3K27 methylation showed a hyper- or hypomethylated DNA configuration due to ITIH5 expression finally leading to re-expression of the tumor suppressor DAPK1. In turn, RNAi-mediated knockdown of DAPK1 in ITIH5-expressing MDA-MB-231 single-cell clones clearly restored cell motility.ConclusionsOur results provide evidence that ITIH5 triggers a reprogramming of breast cancer cells with known stem CSC properties towards an epithelial-like phenotype through global epigenetic changes effecting known tumor suppressor genes like DAPK1. Therewith, ITIH5 may represent an ECM modulator in epithelial breast tissue mediating suppression of tumor initiating cancer cell characteristics which are thought being responsible for the metastasis of breast cancer.

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Epigenetic reprogramming of primary pancreatic cancer cells counteracts their in vivo tumourigenicity.

Publications

Epigenetic reprogramming of primary pancreatic cancer cells counteracts their in vivo tumourigenicity.

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