Reversible Epigenetic Regulation of 14-3-3σ Expression in Acquired Gemcitabine Resistance by Uhrf1 and DNA Methyltransferase 1

Abstract

Although gemcitabine is the most commonly used drug for treating pancreatic cancers, acquired gemcitabine resistance in a substantial number of patients appears to hinder its effectiveness in successful treatment of this dreadful disease. To understand acquired gemcitabine resistance, we generated a gemcitabineresistant pancreatic cancer cell line using stepwise selection and found that, in addition to the known mechanisms of upregulated expression of ribonucleotide reductase, 14-3-3s expression is dramatically upregulated, and that 14-3-3s overexpression contributes to the acquired resistance to gemcitabine and cross-resistance to cytarabine. We also found that the increased 14-3-3s expression in the gemcitabine-resistant cells is due to demethylation of the 14-3-3s gene during gemcitabine selection, which could be partially reversed with removal of the gemcitabine selection pressure. Most importantly, the reversible methylation/ demethylation of the 14-3-3s gene appears to be carried out by DNA methyltransferase 1 under regulation by Uhrf1. These findings suggest that the epigenetic regulation of gene expression may play an important role in gemcitabine resistance, and that epigenetic modification is reversible in response to gemcitabine treatment.

14-3-3σ Contributes to Radioresistance By Regulating DNA Repair and Cell Cycle via PARP1 and CHK2

“…As shown in Figure 4A–B, BxPC-3/Sh-σ had reduced while MiaPaCa-2/σ had increased levels of PARP1 protein and mRNA compared with their respective control cells. Transient 14-3-3σ knockdown in BxPC-3 cells using two different shRNAs or a siRNA pool as describe previously (4) also successfully reduced PARP1 expression (Figure 4C). …”

“…Cell lysate preparation, Western blot analyses, and immunoprecipitation were performed as previously described (4,18). Briefly, cultured cells were harvested, washed with chilled PBS 3 times, and lysed in TNN lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM Na 3 VO 4 , 1 mM DTT, 0.1% SDS, and 2 mM PMSF) for 30 minutes on ice with agitation.…”

The epigenetic integrator UHRF1: on the road to become a universal biomarker for cancer

“…It interacts, among many others, with DNA methyltransferases (DNMTs), proliferating cell nuclear antigen (PCNA), histone deacetylase 1 (HDAC1), ubiquitin specific protease 7 (USP7), euchromatic histone-lysine N methyltransferase 2 (G9a/EHMT2) and Tat Interacting Protein 60 (Tip60) to maintain DNA methylation patterns and histone epigenetic marks in various physiological and pathological conditions [ 18 , 19 , 37 – 42 ]. Together with its partners, UHRF1 ensures the regulation, through “silencing” of a high number of tumor suppressor genes and long non-coding RNAs, including RB1 [ 43 ], p16 ( CDKN2A) [ 44 – 48 ], CDH13 and SHP1 [ 49 ], SOCS3 and 3OST2 [ 50 ], BRCA1 [ 51 ], CDX2, RUNX3, FOXO4, PPARG and PML [ 52 , 53 ], MEG3 [ 54 ] and 14-3-3σ [ 55 ]. Moreover, KISS1 , functioning as a metastasis suppressor in various cancers, also looks to be under the control of UHRF1 [ 56 ].…”

14-3-3σ regulation of and interaction with YAP1 in acquired gemcitabine resistance via promoting ribonucleotide reductase expression

“…It has also been shown previously that increased 14-3-3σ expression associates with poor prognosis of PDAC [ 6 , 7 ]. Recently, using a newly established gemcitabine resistant PDAC cell line, G3K derived from MiaPaCa-2 cells via stepwise gemcitabine selections, we showed that 14-3-3σ expression is upregulated via epigenetic regulation and contributes to the acquired gemcitabine resistance in the gemcitabine-selected PDAC G3K cells [ 8 ]. In addition to the acquired gemcitabine resistance in PDAC cells, 14-3-3σ has been implicated in acquired doxorubicin resistance in breast cancer cells [ 9 ] and in cisplatin resistance of colon cancer cells [ 10 ].…”