ZEB 1‐associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat
2015; Springer Nature; Volume: 7; Issue: 6 Linguagem: Inglês
10.15252/emmm.201404396
ISSN1757-4684
AutoresSimone Meidhof, Simone Brabletz, Waltraut Lehmann, Bogdan‐Tiberius Preca, Kerstin Mock, Manuel Ruh, Julia Schüler, Maria Berthold, Anika M. Weber, Ulrike Burk, Michael Lübbert, Martin Puhr, Zoran Čulig, Ulrich F. Wellner, Tobias Keck, Peter Bronsert, Simon Küsters, Ulrich T. Hopt, Marc P. Stemmler, Thomas Brabletz,
Tópico(s)Cancer-related gene regulation
ResumoResearch Article14 April 2015Open Access Source Data ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat Simone Meidhof Simone Meidhof Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Spemann Graduate School of Biology and Medicine (SGBM), Albert Ludwigs University Freiburg, Freiburg, Germany Faculty of Biology, Albert Ludwigs University Freiburg, Freiburg, Germany Search for more papers by this author Simone Brabletz Simone Brabletz Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Waltraut Lehmann Waltraut Lehmann Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Faculty of Biology, Albert Ludwigs University Freiburg, Freiburg, Germany Search for more papers by this author Bogdan-Tiberius Preca Bogdan-Tiberius Preca Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Faculty of Biology, Albert Ludwigs University Freiburg, Freiburg, Germany Search for more papers by this author Kerstin Mock Kerstin Mock Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Faculty of Biology, Albert Ludwigs University Freiburg, Freiburg, Germany Search for more papers by this author Manuel Ruh Manuel Ruh Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Julia Schüler Julia Schüler Oncotest GmbH, Institute for Experimental Oncology, Freiburg, Germany Search for more papers by this author Maria Berthold Maria Berthold Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Search for more papers by this author Anika Weber Anika Weber Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Search for more papers by this author Ulrike Burk Ulrike Burk Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Search for more papers by this author Michael Lübbert Michael Lübbert Department of Hematology and Oncology, University of Freiburg Medical Center, Freiburg, Germany German Cancer Consortium (DKTK), Freiburg and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Martin Puhr Martin Puhr Division of Experimental Urology, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Zoran Culig Zoran Culig Division of Experimental Urology, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Ulrich Wellner Ulrich Wellner Department of Surgery, University Medical Center Schleswig-Holstein, Campus Lübeck, Germany Search for more papers by this author Tobias Keck Tobias Keck Department of Surgery, University Medical Center Schleswig-Holstein, Campus Lübeck, Germany Search for more papers by this author Peter Bronsert Peter Bronsert Tumorbank Comprehensive Cancer Center Freiburg and Institute of Surgical Pathology, University Medical Center Freiburg, Freiburg, Germany Search for more papers by this author Simon Küsters Simon Küsters Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Search for more papers by this author Ulrich T Hopt Ulrich T Hopt Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Search for more papers by this author Marc P Stemmler Marc P Stemmler Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Thomas Brabletz Corresponding Author Thomas Brabletz Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Erlangen, Germany German Cancer Consortium (DKTK), Freiburg and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Simone Meidhof Simone Meidhof Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Spemann Graduate School of Biology and Medicine (SGBM), Albert Ludwigs University Freiburg, Freiburg, Germany Faculty of Biology, Albert Ludwigs University Freiburg, Freiburg, Germany Search for more papers by this author Simone Brabletz Simone Brabletz Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Waltraut Lehmann Waltraut Lehmann Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Faculty of Biology, Albert Ludwigs University Freiburg, Freiburg, Germany Search for more papers by this author Bogdan-Tiberius Preca Bogdan-Tiberius Preca Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Faculty of Biology, Albert Ludwigs University Freiburg, Freiburg, Germany Search for more papers by this author Kerstin Mock Kerstin Mock Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Faculty of Biology, Albert Ludwigs University Freiburg, Freiburg, Germany Search for more papers by this author Manuel Ruh Manuel Ruh Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Julia Schüler Julia Schüler Oncotest GmbH, Institute for Experimental Oncology, Freiburg, Germany Search for more papers by this author Maria Berthold Maria Berthold Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Search for more papers by this author Anika Weber Anika Weber Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Search for more papers by this author Ulrike Burk Ulrike Burk Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Search for more papers by this author Michael Lübbert Michael Lübbert Department of Hematology and Oncology, University of Freiburg Medical Center, Freiburg, Germany German Cancer Consortium (DKTK), Freiburg and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Martin Puhr Martin Puhr Division of Experimental Urology, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Zoran Culig Zoran Culig Division of Experimental Urology, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Ulrich Wellner Ulrich Wellner Department of Surgery, University Medical Center Schleswig-Holstein, Campus Lübeck, Germany Search for more papers by this author Tobias Keck Tobias Keck Department of Surgery, University Medical Center Schleswig-Holstein, Campus Lübeck, Germany Search for more papers by this author Peter Bronsert Peter Bronsert Tumorbank Comprehensive Cancer Center Freiburg and Institute of Surgical Pathology, University Medical Center Freiburg, Freiburg, Germany Search for more papers by this author Simon Küsters Simon Küsters Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Search for more papers by this author Ulrich T Hopt Ulrich T Hopt Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany Search for more papers by this author Marc P Stemmler Marc P Stemmler Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Thomas Brabletz Corresponding Author Thomas Brabletz Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Erlangen, Germany German Cancer Consortium (DKTK), Freiburg and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Author Information Simone Meidhof1,2,3,‡, Simone Brabletz4,‡, Waltraut Lehmann1,3, Bogdan-Tiberius Preca1,3, Kerstin Mock1,3, Manuel Ruh4, Julia Schüler5, Maria Berthold1, Anika Weber1, Ulrike Burk1, Michael Lübbert6,7, Martin Puhr8, Zoran Culig8, Ulrich Wellner9, Tobias Keck9, Peter Bronsert10, Simon Küsters1, Ulrich T Hopt1, Marc P Stemmler4 and Thomas Brabletz 4,7,11 1Department of General and Visceral Surgery, University of Freiburg Medical Center, Freiburg, Germany 2Spemann Graduate School of Biology and Medicine (SGBM), Albert Ludwigs University Freiburg, Freiburg, Germany 3Faculty of Biology, Albert Ludwigs University Freiburg, Freiburg, Germany 4Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Erlangen, Germany 5Oncotest GmbH, Institute for Experimental Oncology, Freiburg, Germany 6Department of Hematology and Oncology, University of Freiburg Medical Center, Freiburg, Germany 7German Cancer Consortium (DKTK), Freiburg and German Cancer Research Center (DKFZ), Heidelberg, Germany 8Division of Experimental Urology, Innsbruck Medical University, Innsbruck, Austria 9Department of Surgery, University Medical Center Schleswig-Holstein, Campus Lübeck, Germany 10Tumorbank Comprehensive Cancer Center Freiburg and Institute of Surgical Pathology, University Medical Center Freiburg, Freiburg, Germany 11Present address: Chair Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, University Erlangen-Nürnberg, Erlangen, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 9131 8529104; E-mail: [email protected] EMBO Mol Med (2015)7:831-847https://doi.org/10.15252/emmm.201404396 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Therapy resistance is a major clinical problem in cancer medicine and crucial for disease relapse and progression. Therefore, the clinical need to overcome it, particularly for aggressive tumors such as pancreatic cancer, is very high. Aberrant activation of an epithelial–mesenchymal transition (EMT) and an associated cancer stem cell phenotype are considered a major cause of therapy resistance. Particularly, the EMT-activator ZEB1 was shown to confer stemness and resistance. We applied a systematic, stepwise strategy to interfere with ZEB1 function, aiming to overcome drug resistance. This led to the identification of both its target gene miR-203 as a major drug sensitizer and subsequently the class I HDAC inhibitor mocetinostat as epigenetic drug to interfere with ZEB1 function, restore miR-203 expression, repress stemness properties, and induce sensitivity against chemotherapy. Thereby, mocetinostat turned out to be more effective than other HDAC inhibitors, such as SAHA, indicating the relevance of the screening strategy. Our data encourage the application of mechanism-based combinations of selected epigenetic drugs with standard chemotherapy for the rational treatment of aggressive solid tumors, such as pancreatic cancer. Synopsis Therapy resistance is a major problem in cancer medicine. Based on the identification of novel mediators of ZEB1-associated therapy resistance, the HDAC inhibitor mocetinostat is found to efficiently restore drug sensitivity in aggressive cancer cells. Strategy to counteract the well-known cancer-promoting functions of the EMT inducer ZEB1. Identification of the stemness-inhibiting microRNA miR-203 as major ZEB1 target inducing drug sensitivity. Identification of the class I HDAC inhibitor mocetinostat as drug to interfere with ZEB1 function and overcome ZEB1-associated drug resistance. Mocetinostat has better effects in combination with chemotherapeutics compared to other HDACis, such as SAHA. Blueprint for further drug screens with reduction in ZEB1 function as major readout. Introduction Resistance to standard radio- and chemotherapy, as well as to novel targeted therapies, is a major clinical problem in cancer medicine and crucial for disease relapse and progression. Therefore, the clinical need to overcome therapy resistance, particularly for very aggressive tumor types, such as pancreatic cancer, is very high. There are various molecular mechanisms that lead to treatment resistance, and in a general view, many of those have been linked to a stemness-associated survival phenotype (Holohan et al, 2013). Thus, cancer stem cells are considered to be the most resistant fraction of tumor cells, which survive different types of treatment and give rise to tumor recurrence and finally progression toward a multiresistant, often metastatic disease (Clevers, 2011; Borst, 2012). Also, the activation of an epithelial–mesenchymal transition (EMT), considered a driving force toward cancer invasion and metastasis, was associated with treatment resistance (Thiery et al, 2009; Floor et al, 2011). This is of particular relevance, since EMT and stemness were linked at molecular level, explaining why resistant cancer (stem) cells often acquired an undifferentiated EMT phenotype (Polyak & Weinberg, 2009; Singh & Settleman, 2010; Puisieux et al, 2014). The EMT inducer ZEB1 is a transcriptional repressor of epithelial genes, such as E-cadherin and the miR-200 family of microRNAs. ZEB1 and miR-200 members can repress expression of each other in a double-negative feedback loop (Brabletz & Brabletz, 2010). Moreover, since miR-200 as well as miR-203, another microRNA repressed by ZEB1, can also suppress stemness traits, their downregulation by ZEB1 induces an EMT-associated stemness phenotype (Yi et al, 2008; Wellner et al, 2009). Overexpression of ZEB1, as well as subsequent downregulation of miR-200, has already been associated with a pro-survival and drug-resistant phenotype (Mongroo & Rustgi, 2010; Zhang et al, 2015). Furthermore, artificial re-expression of miR-200 family members has been shown to lead to a partial re-sensitization (Buck et al, 2007; Arumugam et al, 2009; Cochrane et al, 2009; Li et al, 2009; Singh et al, 2009; Wellner et al, 2009). How can this knowledge about the molecular links of EMT and drug resistance be translated to clinical application? A depletion of relevant factors, such as ZEB1, selectively in patients' cancer cells is practically impossible. Here, we describe a systematic, stepwise approach to interfere with ZEB1 function and restore drug sensitivity by: (i) identifying additional relevant ZEB1 target genes, (ii) defining ZEB1-dependent epigenetic modifications of these genes, (iii) screening for epigenetic drugs forcing their re-expression, and (v) validating the most promising candidate drug for the restoration of treatment sensitivity. This strategy led to the detection of miR-203 as another important ZEB1 target conferring treatment sensitivity and the identification of the class I HDAC inhibitor mocetinostat, which, in contrast to other HDAC inhibitors such as SAHA, interferes with ZEB1 expression and function and restores sensitivity to chemotherapy. Results miR-203 confers drug sensitivity to ZEB1-expressing, resistant cancer cells EMT and, particularly, the EMT activator ZEB1 are strongly linked to a therapy resistance phenotype (Mongroo & Rustgi, 2010; Zhang et al, 2015). For instance, we have demonstrated that the depletion of ZEB1 in the resistant pancreatic cancer cell line Panc1 results in re-differentiation and re-sensitization to gemcitabine and that a selection for gemcitabine resistance in the sensitive pancreatic cancer cell line BxPC3 induced an EMT phenotype, with high ZEB1 and low E-cadherin expression (Wellner et al, 2009). The same phenotypic changes could be induced by selecting for resistance to docetaxel in the sensitive prostate cancer cell line DU145 (Puhr et al, 2012) and to the EGFR inhibitor Tarceva in the sensitive lung cancer line H358 (Fig 1A). These data indicate that ZEB1 is a crucial determinant for mediating resistance to chemotherapeutics as well as targeted drugs in different cancer types. ZEB1 is a transcriptional repressor, and some of its major target genes, the miR-200 family, have been linked to chemosensitivity. In all cellular systems described here, ZEB1 was upregulated and miR-200 family members were downregulated in the resistant state (Fig 1A). We have previously demonstrated that, like miR-200, the stemness-repressing miR-203 is also a ZEB1 target gene (Wellner et al, 2009), which not only suppresses stemness factors, but also anti-apoptotic factors, such as survivin and BCL-W (Bo et al, 2011; Bian et al, 2012; Wei et al, 2013). Moreover, like miR-200, miR-203 is also downregulated in the resistant state (Fig 1A). These facts prompted us to evaluate miR-203 as a chemosensitizer. Overexpression of miR-200c increased the sensitivity to gemcitabine in the ZEB1-expressing, resistant pancreatic cancer cell lines Panc1 and MiaPaca (Fig 1B and C; Supplementary Fig S1A and Table 1). Strikingly, miR-203 was much more efficient than miR-200c and particularly Panc1 was sensitized to an almost complete growth inhibition. miR-203 also further sensitized the aggressive breast cancer cell line MDA-MB231 to paclitaxel, although the effect was only significant at the EC80 level (Supplementary Fig S1B and Table 1). Figure 1. miR-203 restores drug sensitivity Immunoblots and qRT–PCRs showing that expression levels of miR-203, miR-200, and E-cadherin are increased after ZEB1 knockdown in Panc1, MDA-MB-231. Vice versa, the drug-resistant clones of BxPC3, H358, and DU-145 show increased expression of ZEB1 and decreased expression of the miRNAs and E-cadherin. n = 3, mean ± SEM, except for H358 (data from microarray). Unpaired Student's t-test. Lentiviral overexpression of miR-200c and miR-203 in Panc1 and hPaca1 induces sensitivity to gemcitabine treatment as measured by MTT assay. For the changes in EC50 values, see Table 1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01–0.05, **P = 0.001–0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4). Overexpression of miR-203 decreases expression of the anti-apoptotic factor survivin and sensitizes to gemcitabine-triggered apoptosis as evaluated by cleaved caspase-3 in Western blot and immunofluorescence. Panc1 and hPaca1 were treated with 50 and 5 nM gemcitabine, respectively, for 48 h. Scale bar 20 μm. MTT assay showing increase in gemcitabine resistance after inhibition of endogenous miRNAs in hPaca2 by specific antagomirs against miR-203 or all miR-200 members. For the changes in EC80 values, see Table 1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01–0.05, **P = 0.001–0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4). Overexpression of miR-203 shows reduced numbers of the CD24/CD44 double-positive cancer stem cell population as determined by FACS analysis. The arrow indicates the reduction in the CD24 high subpopulation and reduction in CD133 by miR-203 overexpression in hPaca1 cells. Cancer stem cell sphere assay showing reduced sphere-forming capacity of Panc1 and hPaca1 in miR-203 overexpression cells. Colonies with a diameter greater than 75 μM for Panc1 and greater 30 μM for hPaca1 cells were counted as spheres. n = 3, mean ± SEM, Mann–Whitney U-test. Source data are available online for this figure. Source Data for Figure 1 [emmm201404396-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Table 1. Shift in EC50 (EC80) by microRNAs or antagomirs Cell line miRNA Drug EC50 (nM) Panc1 ctrl Gemcitabine > 10,000 miR-200 Gemcitabine 43 miR-203 Gemcitabine 19 MiaPaca ctrl Gemcitabine 830 miR-200 Gemcitabine 22 miR-203 Gemcitabine 23 EC50/EC80 hPaca 1 ctrl Gemcitabine 10.8/98 miR-200 Gemcitabine 8.7/28 miR-203 Gemcitabine 5.2/9.4 hPaca 2 ctrl Gemcitabine 6.2/34 a miR-200 Gemcitabine 8.2/88 a miR-203 Gemcitabine 7.6/85 MDA-MB231 ctrl Paclitaxel 4.9/1050 miR-200 Paclitaxel 6.1/24 miR-203 Paclitaxel 4.4/15 To validate the results in clinically more relevant settings, we isolated cancer cells from patient-derived pancreatic adenocarcinomas and selected two representative cases. hPaca1 has an undifferentiated phenotype similar to Panc1, with high ZEB1 and low E-cadherin, miR-200, and miR-203 expression, whereas hPaca2 is more differentiated with an inverse expression pattern, similar to BxPC3 (Supplementary Fig S2A). Moreover, like Panc1, hPaca1 has a CD24high/44high subpopulation, considered to exert a tumorigenic stemness phenotype (Supplementary Fig S2B). Of note, hPaca2 is almost completely lacking this subpopulation. Compared with Panc1, both lines showed a higher, but similar sensitivity to gemcitabine (Supplementary Fig S2C). Nevertheless, the ZEB1-expressing line hPaca1 could be further sensitized to even very low gemcitabine doses by miR-203 and miR-200c overexpression. This effect was most significant at the EC80 level (Fig 1B and Table 1). A reverse strategy was applied for the differentiated line hPaca2, which already expresses miR-200 and miR-203. A combination of antagomirs against the endogenously expressed five miR-200 family members reduced gemcitabine sensitivity, even at high doses, again with the most significant effects reached at EC80 levels (Fig 1D and Table 1). Antagomir treatment against miR-203 alone had the same effect. These data underscore the role of miR-203 as inducer of drug sensitivity, which can be partially explained by a pro-apoptotic (Fig 1C and Supplementary Fig S1A and B) and stemness-repressing function, as indicated by the reduction in the cancer-stem-cell-associated markers CD24high/44high and CD133 (Fig 1E), as well as the sphere-forming capacity (Fig 1F). Although overexpression of miR-203 alone even enhanced the proliferative capacity, it induced (Panc1) or slightly enhanced (hPaca1) anti-proliferative effects if combined with gemcitabine (Supplementary Fig S1C). Identification of ZEB1-dependent epigenetic modifications on its target genes We aimed to interfere with ZEB1 function by forcing a re-expression of its silenced, drug-sensitizing target genes. To this end, we first determined epigenetic modifications conferred by the transcriptional repressor ZEB1 on miR-200, miR-203, and E-cadherin genes by comparing control and ZEB1 knockdown samples of the aggressive cell lines Panc1 and MDA-MB231. ZEB1 depletion induced an increase in the active histone marks H3K4me3, H3ac, H4ac, and H3K9ac in all gene loci, besides the miR-200a,b, 429 cluster (Fig 2A and B). In addition, a depletion of ZEB1 resulted in a decrease in the repressive histone mark H3K27me3 on the miR-203 locus. DNA methylation patterns were also related to the ZEB1 expression status in MDA-MB231, where CpG islands in the loci of miR-203 and the miR-200c/miR-141 cluster were methylated and ZEB1 depletion resulted in an almost complete demethylation. DNA methylation patterns in Panc1 were inconsistent and not associated with the ZEB1 expression status. The ZEB1-related epigenetic changes could be verified again in the reverse setting using the drug-sensitive line BxPC3, lacking ZEB1 and expressing endogenous miR-200, miR-203, and E-cadherin (Fig 2C). Here, the loci were demethylated, but the selection of drug-resistant, ZEB1-expressing clones induced a complete methylation and a reduction in active histone marks. Figure 2. ZEB1-dependent epigenetic modifications A. Schemes for the genomic loci of the miRNA and E-cadherin genes, showing regions of the CpG islands (yellow), of the qRT–PCR amplicon for chromatin immunoprecipitation (ChIP) analysis (blue) and of the bisulfite sequencing (red). B, C. Histone marks were analyzed using ChIP coupled to qRT–PCR for Panc1 control versus shZEB, MDA-MB-231 control versus shZEB (B), and BxPC3 control versus gemcitabine resistant (gr) (C). In MDA-MB-231 and Panc1, the active histone marks H3K4me3, H3ac, H4ac, and H3K9ac were enriched. Vice versa, in the drug-resistant clones of BxPC3, the active marks were reduced in the CpG islands. The repressive histone mark H3K27me3 was not detectable in the miR-200 loci, but in the loci of miR-203 and E-cadherin in Panc1 and MDA-MB-231. DNA methylation status was determined by bisulfite sequencing. Depletion of ZEB1 in MDA-MB-231 resulted in almost complete demethylation, whereas the selection of drug-resistant, ZEB1-expressing clones in BxPC3 induced complete methylation. n = 2 (Panc1) or 3 (MDA-MB-231 and BxPC3), mean ± SEM; unpaired Student's t-test. Download figure Download PowerPoint Screening for epigenetic drugs interfering with ZEB1 function We next applied a screening strategy for epigenetic drugs by selecting for their ability to re-activate expression of silenced ZEB1 target genes. Re-expression of miR-203 was used as the major readout. The best candidate(s) should then be tested for sensitizing cancer cells to chemotherapy. Based on the detected ZEB1-dependent epigenetic modifications (Fig 2) as well as the known co-repressors of ZEB1 (Wang et al, 2007, 2009; Aghdassi et al, 2012; Gheldof et al, 2012), we focused on inhibitors of histone deacetylases (HDACs), the lysine-specific demethylase 1 (LSD1), polycomb repressor complex 2 (PRC2) factors, and DNA methyltransferases (DNMT). Single-agent treatment with the LSD1 inhibitor TCP, the PRC2 complex inhibitors DZnep, ad dia, and cAra and the DNMT inhibitor dAza in ZEB1-expressing lines Panc1 and hPaca1 did not consistently re-activate expression of silenced miR-203 and miR-200 members (Fig 3A, Supplementary Fig S2D and E and Supplementary Table S1 for statistical significance). We further concentrated on HDAC inhibitors, since HDAC1 and HDAC2 are known ZEB1 co-repressors (Wang et al, 2009; Aghdassi et al, 2012) and the most prominent ZEB1-dependent epigenetic modifications we detected are conducted by HDACs (Fig 2). SAHA (vorinostat) did not or only weakly activate expression of the microRNAs (Fig 3B and Supplementary Fig S2E and F). Trichostatin A, despite upregulating expression of miR-203, also led to an increase in ZEB1 in Panc1, indicating an unspecific stress reaction or gene activation induced by the applied drug doses (Fig 3B and Supplementary Fig S2F). Entinostat (MS-275) and mocetinostat (MGCD0103) led to a consistent upregulation of the microRNAs, in particular of silenced miR-203. In particular, mocetinostat treatment not only strongly upregulated miR-203, but also reduced expression of ZEB1 on both mRNA and protein level (Fig 3B and C, Supplementary Figs S2F and S3A). We therefore focused on this drug, which has the highest specificity for HDAC1 (Fournel et al, 2008). Mocetinostat induced a global increase in H3 and H4 acetylation, without changing the expression of HDACs (Fig 3C and Supplementary Fig S3A) and an increase in the active histone marks H3ac, H4ac, H3K9ac, and H3K4me3 at ZEB1 target gene loci (Fig 3D and Supplementary Fig S3B). Mocetinostat also conferred miR-203-related functions affecting drug resistance, such as suppression of survivin expression and suppression of stemness properties in Panc1 and hPaca1 (Fig 3E and Supplementary Fig S3C). Notably, mocetinostat had no effect in the differentiated patient-derived line hPaca2, already expressing high miR-203 and low ZEB1, compared to its counterpart hPaca1 (Supplementary Fig S3A). Figure 3. Screening of epigenetic drugs for upregulation of miRNAs and downregulation of ZEB1 Heat map showing the relative expression levels after drug treatment for 48 h in Panc1. Values measured by qRT–PCR were depicted with the software GENE-E. Only mocetinostat upregulated the miRNAs and downregulated ZEB1. Relative expression of indicated genes in Panc1 measured by qRT–PCR after treatment with different HDAC inhibitors. Note the downregulation of ZEB1 and upregulation of miR-203, miR-200, and E-cadherin by mocetinostat. n = 3, mean ± SEM; unpaired Student's t-test. For significance, see Supplementary Table S1. Immunoblot and immunofluorescence showing that mocetinostat treatment (1 μM, 48 h) reduced ZEB1 expression and induced E-cadherin in Panc1. Expression of histone deacetylases was not altered by mocetinostat, but histone acetylation was induced. Scale bar 10 μm. Chromatin immunoprecipitation analysis validated mocetinostat-induced (1 μM, 48 h) enrichment of the active histone marks H3ac, H4ac, H3K9ac, and H3K4me3 at ZEB1 target gene loci in Panc1. n = 3, mean ± SEM; unpaired Student's t-test. Mocetinostat treatment reduced expression of the anti-apoptotic miR-203 target survivin and sphere-forming capacity in Panc1 when pre-treated with mocetinostat for 48 h. n = 3, mean ± SEM; Mann–Whitney U-test. Source data are available online for this figure. Source Data for Figure 3 [emmm201404396-sup-0003-SDataFig3.pdf] Download figure Download PowerPoint The class I HDAC inhibitor mocetinostat restores drug sensitivity The effects of mocetinostat on two regulators of drug resistance—downregulation of ZEB1 expression and upregulation of miR-203—prompted us to investigate whether this substance can restore drug sensitivity. We now largely focused on pancreatic cancer, since this tumor has a particular poor prognosis and treatment options are rare
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