DNA methylation is thought to induce transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators unable to bind their target sites when methylated, and the recruitment of transcriptional repressors with specific affinity for methylated DNA. The Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. Here, we present MBD2 ChIPseq data obtained from the endogenous MBD2 in an isogenic cellular model of oncogenic transformation of human mammary cells. In immortalized (HMEC-hTERT) or transformed (HMLER) cells, MBD2 was found in a large proportion of methylated regions and associated with transcriptional silencing. A redistribution of MBD2 on methylated DNA occurred during oncogenic transformation, frequently independently of local DNA methylation changes. Genes downregulated during HMEC-hTERT transformation preferentially gained MBD2 on their promoter. Furthermore, depletion of MBD2 induced an upregulation of MBD2-bound genes methylated at their promoter regions, in HMLER cells. Among the 3,160 genes downregulated in transformed cells, 380 genes were methylated at their promoter regions in both cell lines, specifically associated by MBD2 in HMLER cells, and upregulated upon MBD2 depletion in HMLER. The transcriptional MBD2-dependent downregulation occurring during oncogenic transformation was also observed in two additional models of mammary cell transformation. Thus, the dynamics of MBD2 deposition across methylated DNA regions was associated with the oncogenic transformation of human mammary cells.
ABSTRACT Patients with colorectal cancer (CRC) frequently develop liver metastases during the course of their disease. A substantial proportion of them receive neoadjuvant FOLFOX (5-Fluorouracil, Oxaliplatin, Leucovorin) prior to surgery in an attempt to enable successful surgical removal of their metastases and to reduce the risk of recurrence. Yet, the majority of patients progress during treatment or recur following surgery, and molecular mechanisms that contribute to FOLFOX resistance remain poorly understood. Here, using a combination of phenotypic, transcriptomic and genomic analyses of both tumor samples derived from patients with metastatic CRC and matching patient-derived tumor organoids (PDTOs), we characterize a novel FOLFOX resistance mechanism and identify inhibitors that target this mechanism to resensitize metastatic organoids to FOLFOX. Resistant PDTOs, identified after in vitro exposure to FOLFOX, exhibited elevated expression of E2F pathway, S phase, G 2 /M and spindle assembly checkpoints (SAC) genes. Similar molecular features were detected in CRLM from patients with progressive disease while under neoadjuvant FOLFOX treatment, highlighting the relevance of this finding. FOLFOX resistant PDTOs displayed inactivating mutations of TP53 and exhibited transcriptional features of P53 pathway downregulation. We found that they accumulated in early S-phase and underwent significant DNA damage during FOLFOX exposure, thereafter arresting in G 2 /M while they repaired their DNA after FOLFOX withdrawal. In parallel, results of a large kinase inhibitor screen indicated that drugs targeting regulators of the DNA damage response, G 2 M checkpoint and SAC had cytotoxic effects on PDTOs generated from patients whose disease progressed during treatment with FOLFOX. Corroborating this finding, CHK1 and WEE1 inhibitors were found to synergize with FOLFOX and sensitize previously resistant PDTOs. Additionally, targeting the SAC master regulator MPS1 using empesertib after exposure to FOLFOX, when cells accumulate in G 2 M, was also very effective to kill FOLFOX-resistant PDTOs. Our results indicate that targeted and timely inhibition of specific cell cycle checkpoints shows great potential to improve response rates to FOLFOX in patients with metastatic CRC, for whom therapeutic alternatives remain extremely limited.
Abstract Breast cancer-associated fibroblasts (CAFs) have a crucial role in tumor initiation, metastasis and therapeutic resistance by secreting various growth factors, cytokines, protease and extracellular matrix components. Soluble factors secreted by CAFs are involved in many pathways including inflammation, metabolism, proliferation and epigenetic modulation, suggesting that CAF-dependent reprograming of cancer cells affects a large set of genes. This paracrine signaling has an important role in tumor progression, thus deciphering some of these processes could lead to relevant discoveries with subsequent clinical implications. Here, we investigated the mechanisms underlying the changes in gene expression patterns associated with the cross-talk between breast cancer cells and the stroma. From RNAseq data obtained from breast cancer cell lines grown in presence of CAF-secreted factors, we identified 372 upregulated genes, exhibiting an expression level positively correlated with the stromal content of breast cancer specimens. Furthermore, we observed that gene expression changes were not mediated through significant DNA methylation changes. Nevertheless, CAF-secreted factors but also stromal content of the tumors remarkably activated specific genes characterized by a DNA methylation pattern: hypermethylation at transcription start site and shore regions. Experimental approaches (inhibition of DNA methylation, knockdown of methyl-CpG-binding domain protein 2 and chromatin immunoprecipitation assays) indicated that this set of genes was epigenetically controlled. These data elucidate the importance of epigenetics marks in the cancer cell reprogramming induced by stromal cell and indicated that the interpreters of the DNA methylation signal have a major role in the response of the cancer cells to the microenvironment.
Geno- and phenotypic heterogeneity amongst cancer cell subpopulations are established drivers of treatment resistance and tumour recurrence. However, due to the technical difficulty associated with studying such intra-tumoural heterogeneity, this phenomenon is seldom interrogated in conventional cell culture models. Here, we employ a fluorescent lineage technique termed "optical barcoding" (OBC) to perform simultaneous longitudinal tracking of spatio-temporal fate in 64 patient-derived colorectal cancer subclones. To do so, patient-derived cancer cell lines and organoids were labelled with discrete combinations of reporter constructs, stably integrated into the genome and thus passed on from the founder cell to all its clonal descendants. This strategy enables the longitudinal monitoring of individual cell lineages based upon their unique optical barcodes. By designing a novel panel of six fluorescent proteins, the maximum theoretical subpopulation resolution of 64 discriminable subpopulations was achieved, greatly improving throughput compared with previous studies. We demonstrate that all subpopulations can be purified from complex clonal mixtures via flow cytometry, permitting the downstream isolation and analysis of any lineages of interest. Moreover, we outline an optimized imaging protocol that can be used to image optical barcodes in real-time, allowing for clonal dynamics to be resolved in live cells. In contrast with the limited intra-tumour heterogeneity observed in conventional 2D cell lines, the OBC technique was successfully used to quantify dynamic clonal expansions and contractions in 3D patient-derived organoids, which were previously demonstrated to better recapitulate the heterogeneity of their parental tumour material. In summary, we present OBC as a user-friendly, inexpensive, and high-throughput technique for monitoring intra-tumoural heterogeneity in in vitro cell culture models.
Dans certains cancers, le ligand netrine-1 (NTN1) est surexprime, induisant alors une inhibition de l'apoptose induite par ses recepteurs a dependance DCC et UNC5H, et promouvant ainsi la progression tumorale. Neanmoins, dans d'autres cancers, l'inhibition selective de l'apoptose induite par cette voie de signalisation est liee a la perte d'expression de proteines pro apoptotiques effectrices. Dans cette etude, nous avons montre chez l'homme qu'une forte proportion de cancers mammaires et pulmonaires subit des pertes d'expression de DAPK1 et NTN1 liees a l'hypermethylation de leur promoteur. DAPK1 est une proteine kinase notamment responsable de la transmission du signal proapoptotique des recepteurs a dependance en absence de leur ligand netrine-1. L'inhibition de la methylation de l'ADN a l'aide d'epidrogues telles que la decitabine dans les cancers exprimant faiblement NTN1 restaure l'expression a la fois de DAPK1 et NTN1. Ainsi, la combinaison de traitements decitabine avec des strategies induisant le silence transcriptionnel de NTN1, ou des anticorps bloquant l'interaction entre le ligand (NTN1) et son recepteur (UNC5B dans le cas present), induit la potentialisation de la mort cellulaire des tumeurs, et bloque efficacement la croissance tumorale dans differents modeles animaux. Ainsi, combiner l'utilisation d'inhibiteurs de la methylation de l'ADN avec des agents neutralisant la netrine-1 pourrait representer une strategie pertinente pour combattre ces cancers
Research Article4 July 2016Open Access Transparent process Inhibition of DNA methylation promotes breast tumor sensitivity to netrin-1 interference Mélodie Grandin Mélodie Grandin Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Pauline Mathot Pauline Mathot Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Guillaume Devailly Guillaume Devailly Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Yannick Bidet Yannick Bidet Laboratoire d'Oncologie Moléculaire, Centre Jean Perrin, Clermont-Ferrand, France Search for more papers by this author Akram Ghantous Akram Ghantous Epigenetics Group, IARC, Lyon, France Search for more papers by this author Clementine Favrot Clementine Favrot Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Benjamin Gibert Benjamin Gibert Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Nicolas Gadot Nicolas Gadot Endocrine Differentiation Laboratory, CRCL, Université de Lyon, Hospices Civils de Lyon, Hôpital Edouard Herriot, Anatomie Pathologique, Lyon, France Search for more papers by this author Isabelle Puisieux Isabelle Puisieux Targeting of the tumor and its immune environment Laboratory CRCL, INSERM U1052, CNRS UMR5286, UCBL, CLB, Lyon, France Search for more papers by this author Zdenko Herceg Zdenko Herceg Epigenetics Group, IARC, Lyon, France Search for more papers by this author Jean-Guy Delcros Jean-Guy Delcros Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Agnès Bernet Agnès Bernet Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Patrick Mehlen Corresponding Author Patrick Mehlen orcid.org/0000-0001-8070-7695 Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Robert Dante Corresponding Author Robert Dante Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Mélodie Grandin Mélodie Grandin Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Pauline Mathot Pauline Mathot Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Guillaume Devailly Guillaume Devailly Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Yannick Bidet Yannick Bidet Laboratoire d'Oncologie Moléculaire, Centre Jean Perrin, Clermont-Ferrand, France Search for more papers by this author Akram Ghantous Akram Ghantous Epigenetics Group, IARC, Lyon, France Search for more papers by this author Clementine Favrot Clementine Favrot Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Benjamin Gibert Benjamin Gibert Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Nicolas Gadot Nicolas Gadot Endocrine Differentiation Laboratory, CRCL, Université de Lyon, Hospices Civils de Lyon, Hôpital Edouard Herriot, Anatomie Pathologique, Lyon, France Search for more papers by this author Isabelle Puisieux Isabelle Puisieux Targeting of the tumor and its immune environment Laboratory CRCL, INSERM U1052, CNRS UMR5286, UCBL, CLB, Lyon, France Search for more papers by this author Zdenko Herceg Zdenko Herceg Epigenetics Group, IARC, Lyon, France Search for more papers by this author Jean-Guy Delcros Jean-Guy Delcros Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Agnès Bernet Agnès Bernet Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Patrick Mehlen Corresponding Author Patrick Mehlen orcid.org/0000-0001-8070-7695 Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Robert Dante Corresponding Author Robert Dante Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France Search for more papers by this author Author Information Mélodie Grandin1, Pauline Mathot1, Guillaume Devailly1, Yannick Bidet2, Akram Ghantous3, Clementine Favrot1, Benjamin Gibert1, Nicolas Gadot4, Isabelle Puisieux5, Zdenko Herceg3, Jean-Guy Delcros1, Agnès Bernet1, Patrick Mehlen 1,‡ and Robert Dante 1,‡ 1Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France 2Laboratoire d'Oncologie Moléculaire, Centre Jean Perrin, Clermont-Ferrand, France 3Epigenetics Group, IARC, Lyon, France 4Endocrine Differentiation Laboratory, CRCL, Université de Lyon, Hospices Civils de Lyon, Hôpital Edouard Herriot, Anatomie Pathologique, Lyon, France 5Targeting of the tumor and its immune environment Laboratory CRCL, INSERM U1052, CNRS UMR5286, UCBL, CLB, Lyon, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 4 7878 2870; E-mail: [email protected] *Corresponding author. Tel: +33 7 7878 5922; E-mail: [email protected] EMBO Mol Med (2016)8:863-877https://doi.org/10.15252/emmm.201505945 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 In a number of human cancers, NTN1 upregulation inhibits apoptosis induced by its so-called dependence receptors DCC and UNC5H, thus promoting tumor progression. In other cancers however, the selective inhibition of this dependence receptor death pathway relies on the silencing of pro-apoptotic effector proteins. We show here that a substantial fraction of human breast tumors exhibits simultaneous DNA methylation-dependent loss of expression of NTN1 and of DAPK1, a serine threonine kinase known to transduce the netrin-1 dependence receptor pro-apoptotic pathway. The inhibition of DNA methylation by drugs such as decitabine restores the expression of both NTN1 and DAPK1 in netrin-1-low cancer cells. Furthermore, a combination of decitabine with NTN1 silencing strategies or with an anti-netrin-1 neutralizing antibody potentiates tumor cell death and efficiently blocks tumor growth in different animal models. Thus, combining DNA methylation inhibitors with netrin-1 neutralizing agents may be a valuable strategy for combating cancer. Synopsis Inhibition of DNA methylation with decitabine restores expression of netrin-1 (NTN1) and DAPK1 in human breast tumors. Combining decitabine with NTN1 neutralization potentiates tumor cell death and blocks tumor growth in various animal models. NTN1 and DAPK1 are frequently hypermethylated and lost in human breast cancers. NTN1 and DAPK1 loss of expression is associated with a loss of apoptosis induction upon netrin-1 targeting antibody treatment. Epigenetic drugs such as decitabine (DAC) induce demethylation and upregulation of DAPK1 and NTN1 in tumor cells, thus leading to cell resensitization to netrin-1 targeting antibodies. Combinatorial DAC + net1-mAb treatment triggers cell death in vitro and tumor growth inhibition in mice. Introduction Recent research focusing on a specific functional family of receptors, namely the dependence receptors (DRs) (Mehlen et al, 1998; Llambi et al, 2001), has highlighted their implication in inhibiting tumor progression. Indeed, in contrast to most cellular receptors, a dual role characterizes these transmembrane receptors: in the presence of their respective ligands, they provide a typical positive signal (promoting cell survival, migration, proliferation, etc.), while the absence of ligand triggers a cascade of signaling events leading to apoptotic cell death. The DR protein family currently contains approximately twenty known members, and one of their most exhaustively studied ligands is Netrin-1. This DR ligand was the first secreted attractive axon guidance cue to be described in the early nineties, since then, the role of netrin-1 in many biological functions has been established (Cirulli & Yebra, 2007; Mehlen & Guenebeaud, 2010; Ramkhelawon et al, 2014). Netrin-1 receptors, namely, are the Deleted in Colorectal Carcinoma (DCC), the Uncoordinated-5-Homologs (UNC5H1-4/A–D). The ability of these receptors to trigger apoptosis in settings under ligand-limited conditions was shown to be a constrain for impede tumor progression. Indeed, the inactivation of UNC5C, and DCC, or the mutation of the DCC-inducing apoptosis domain is associated with tumor development and progression in mouse models (Bernet et al, 2007; Castets et al, 2012; Krimpenfort et al, 2012). Furthermore, in line with these observations, DCC and UNC5H are silenced in many cancers, either by loss of heterozygosity or epigenetic mechanisms (Hedrick et al, 1994; Bernet et al, 2007; Shin et al, 2007). Alternatively, in some types of cancers, an upregulation of NTN1 provides a similar tumor growth selective advantage by abolishing their dependency on netrin-1 availability in the micro-environment (Fitamant et al, 2008). This upregulation is particularly marked in some cases of aggressive breast cancer and in breast metastasis (Fitamant et al, 2008; Harter et al, 2014). The reliance on netrin-1 for tumor growth was rapidly perceived as an opportunity for therapeutic intervention, since it was speculated that the disruption of the binding of netrin-1 to its receptors should induce apoptotic cell death in vitro and tumor growth inhibition in vivo. Consistently, the silencing of netrin-1 or the development of biological agents interfering with netrin-1/DRs interactions has been shown to efficiently reduce tumor growth and metastasis in different animal models (Fitamant et al, 2008; Delloye-Bourgeois et al, 2009a,b; Paradisi et al, 2013; Grandin et al, 2016). Moreover, an anti-netrin-1 antibody is under preclinical evaluation and should be assessed in early clinical trials in 2016. Nevertheless, a substantial fraction of human tumors appears to conserve the expression of netrin-1 receptors without upregulating NTN1 expression, suggesting that the downstream DR pathways may be impaired (Shin et al, 2007; Mian et al, 2011; Krimpenfort et al, 2012). In cancers, epigenetic modifications are frequently associated with an increase in the expression of anti-apoptotic proteins and with the inactivation of factors inducing apoptosis (Baylin & Ohm, 2006). In this context, previous studies have reported that DAPK1, a serine threonine kinase responsible for UNC5H-induced apoptosis, is downregulated in various cancers (Llambi et al, 2005; Guenebeaud et al, 2010). Furthermore, mechanistic analyses have demonstrated a direct relationship between the hypermethylation of the CpG island (CGi) located within the DAPK1 promoter region and its downregulation (Raval et al, 2007; Pulling et al, 2009; Mian et al, 2011; Kilinc et al, 2012). Finally, it was shown that treatment with decitabine (5-aza-2′-deoxycytidine, DAC) inhibits the DNA methylation of the DAPK1 promoter and restores DAPK1 expression in lung cancer cell lines (Tang et al, 2004). Inhibition of DNA methylation may prove to be a promising approach for combating cancer (Yang et al, 2010; Rodriguez-Paredes & Esteller, 2011a; Dawson & Kouzarides, 2012; Tsai et al, 2012). Concordantly, DAC showed anti-leukemic effects in several clinical trials and has been approved by FDA for the treatment of myelodysplastic syndromes and recent studies indicated that DAC shows some effects in preclinical models (Tsai et al, 2012). Here, we report that treatment with DAC restores the pro-apoptotic machinery linked to the netrin-1/DR-mediated cell death signaling pathway in breast cancer cell lines and patient-derived xenografts and sensitize cancer cells to netrin-1 neutralizing agents. We thus provide evidence that combining DNA methylation inhibitors with a netrin-1 neutralizing antibody empowers tumor cell death in vitro and tumor growth inhibition in mice. Results Epigenetic downregulation of netrin-1 is associated with the epigenetic downregulation of DAPK1, in human breast cancers In order to study the mechanisms underlying inhibition of netrin-1/DRs-mediated apoptosis, which in a fraction of tumors is neither dependent on netrin-1 upregulation nor DR silencing, we investigated DAPK1 expression and DNA methylation in breast tumors. We thus examined differentially methylated regions (DMRs) associated with malignant transformation in breast cancer samples (n = 92) available from The Cancer Genome Atlas (TCGA) by comparing paired normal/tumoral data obtained from the HumanMethylation450K Array (HM450, Illumina) and by high-throughput sequencing of polyadenylated RNA (RNAseq). This comparison focused on the level of DNA methylation within the 5′-end of NTN1 (position −765 to + 1,300 relative to the transcription start site, TSS) and DAPK1 (+ 365 and + 838 relative to the TSS) and revealed that these regions were hypermethylated (threshold = 2) in about 30% of tumoral samples compared with their normal counterparts (Fig 1A and B). Furthermore, NTN1 was downregulated (Fig 1C, fold change (FC) ≤ 0.5) in 43% of cases and the "NTN1-low" samples were hypermethylated (Fig 1G; P = 3 × 10−2, two-sided Mann–Whitney test) when compared with samples exhibiting no NTN1 downregulation (FC ≥ 1.3). Using the same approach for DAPK1, we observed that samples exhibiting DAPK1 downregulation (29% of the samples, Fig 1D) were also hypermethylated (P = 3 × 10−4) when compared with the other samples (Fig 1H). These epigenetic modifications did not seem to be independent events since in NTN1-hypermethylated samples (n = 23, FC ≥ 2), DAPK1 was also hypermethylated (mean FC = 2.22), while in NTN1-hypomethylated samples (n = 13, FC < 0.7), DAPK1 was not hypermethylated (mean FC = 0.93). The relationship between DAPK1/NTN1 downregulation and DNA hypermethylation was also observed in a larger number of breast cancer samples (n = 807) available on TCGA data portal. Indeed, the mean percentage of CpG methylation (Fig 1E and F) of DAPK1 and NTN1 were inversely correlated with their levels of expression (Pearson's r = −0.32, P < 10−18 and Pearson's r = −0.14, P = 6.7 × 10−5, respectively), suggesting that DNA methylation represses DAPK1 and NTN1 transcription in human breast tumors. Figure 1. NTN1 and DAPK1 are hypermethylated and downregulated in human breast cancers A, B. DNA methylation level of NTN1 (A) and DAPK1 (B) 5′ regions (Illumina's HumanMethylation450K Array (HM450) from The Cancer Genome Atlas breast cohort) in paired breast tissues (normal: green circles, tumor: red circles), n = 92, Wilcoxon matched-pairs signed rank test, P = 0.004 and P = 0.0008. C, D. NTN1 (D) and DAPK1 (G) gene expression in paired breast tissues (normal: green bars, tumor: red bars), RNAseq from TCGA breast cohort, n = 112 and 114, respectively. E, F. Correlation between NTN1 (E) and DAPK1 (F) gene expression and DNA methylation in the breast cancer cohort (TCGA, n = 807). Pearson correlation, P = 6.7.10−5, r = −0.14 for NTN1 (A) and P < 10−16, r = −0.32 for DAPK1 (B), respectively. G, H. Tumor/normal DNA methylation ratio of NTN1 (G) and DAPK1 (H) in human breast tumors (data extracted from TCGA cohort, paired samples) according to gene expression (downregulated FC ≤ 0.5, down, n = 33, or upregulated FC ≥ 1.3, up, n = 16). P = 3 × 10−2 and P = 3 × 10−4 two-sided Mann–Whitney test, for NTN1 and DAPK1, respectively. Download figure Download PowerPoint Although we cannot excluded a bias due to stromal contamination, it should be noted that, in paired samples, while hypermethylation was observed in tumoral tissues, very low levels of DNA methylation were measured in their normal counterparts (Fig 1A and B). For both genes, a region (not represented on HM450) located at the 3′-end of the CGis (Fig 2A, light gray boxes) was selected for the quantitative analysis of DNA methylation of breast cancer biopsies (tumor bank—Centre Léon Bérard) by bisulfite pyrosequencing. The analysis indicated that the mean percentage of CpG methylation of the DAPK1 and NTN1 pyrosequenced regions were inversely correlated (Pearson's r = −0.66, P = 0.003, and Pearson's r = −0.55, P = 0.008, respectively) with their levels of expression (Fig EV1A and B). Altogether, these data suggested that DNA methylation is involved in the downregulation of NTN1 and DAPK1 in human breast cancers. Figure 2. DNA methylation and demethylation in mammary cell lines A. Methyl-Cap-seq read density profiles of the 5′ end of DAPK1, UNC5B, and NTN1 in MDA-MB-231 (blue) and HMLER (green) cells. Red boxes represent the CpG islands (CGis); light gray boxes the regions analyzed by bisulfite PCR sequencing; dark gray boxes represent the regions analyzed by parallel sequencing of amplicons from bisulfite modified DNA; and black boxes represent the exons and UTR. Chromosome coordinates of each gene are given (black lines). B, C. Gene expression was measured by qRT–PCR after 72 h for MDA-MB-231 (B) and HMLER cells (C) treated daily with DAC (10 μM). PBGD expression level was used as an internal control. Data are expressed as mean ± s.e.m. of at least 3 independent experiments. ****P < 0.0001, two-tailed unpaired Student's t-test. D, E. Measurement of DNA hypomethylation of the DAPK1 (D) and NTN1 (E) promoters after decitabine treatment of MDA-MB-231 and HMLER cells. Over 1960 sequences were analyzed per group in 2 independent experiments. ****P < 0.0001, two-way ANOVA and post hoc Tukey test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. NTN1 and DAPK1 are concomitantly altered in human breast cancers Bisulfite PCR sequencing (4 CpGs analyzed, region: light gray boxes in Fig 1A) indicated that DNA methylation of the NTN1 CpG island (CGi) was inversely correlated with its expression. Pearson correlation, P = 0.008, r = −0.55, n = 18. Bisulfite PCR sequencing (3 CpGs analyzed, region: light gray boxes in Fig 2A) indicated that DNA methylation of the DAPK1 CGi was inversely correlated with its expression. Pearson correlation, P = 0.003, r = −0.66, n = 22. Tissue microarrays (70 paraffin embedded sections) from human breast carcinomas were immuno-stained with antibodies against DAPK1, UNC5B, and netrin-1. Samples were classified in quartiles according to the level of netrin-1 expression. The levels of DAPK1 and UNC5B expression (index constructed from the percentage of sections exhibiting a positive staining) in the first and fourth quartiles of netrin-1 expressing groups were compared using a chi-squared test. Representative staining corresponding to low and high levels of expression is shown for each antibody, and expression levels were determined from the percentage of sections exhibiting a positive staining. As a control, staining of the samples using a non-related isotype antibody was performed. Quantification of the presence or absence of alterations in gene A was associated with the presence or absence of alterations in gene B in human breast tumors which was determined from cBioPortal web site, z-score threshold ± 2.2 Individual methylation plots. Red rectangles represent methylated CpG and blue rectangles unmethylated CpG. Mean DNA methylation inhibition of DAPK1 and NTN1 in MDA-MB-231 and HMLER cells upon DAC treatment. Download figure Download PowerPoint To determine whether this concomitant change in DAPK1 and NTN1 expression was also observed at the protein levels, DAPK1, UNC5B, and netrin-1 were measured by immunohistochemistry (IHC) using tissue microarrays (70 sections) from human breast ductal carcinoma (Super Bio Chips). This analysis revealed, that, netrin-1-low samples also exhibited low levels of DAPK1 (χ2 test, P = 0.04). In contrast, UNC5B levels were the same, irrespective of netrin-1 levels (Fig EV1C and D). This result was validated in the TCGA cohort of breast tumors samples (n = 807), where a correlation between DAPK1 and NTN1 expression was observed (odd ratio = 4.71, P = 0.03, Fig EV1E). In order to establish an in vitro experimental model mimicking the DNA methylation alterations observed in breast cancer tissues, DNA methylation patterns of two cancer cell lines were determined by parallel sequencing. Pull-down assays were conducted using the MDA-MB-231 cell line derived from human breast cancer, and the HMLER cell line generated through the in vitro transformation of human mammary cells (Elenbaas et al, 2001; Morel et al, 2008). Methylated DNA fragments were selected using a recombinant protein containing the Methyl-CpG-binding domain of MBD2 of MBD2 (Methyl-Cap-seq), from MDA-MB-231 cell line derived from human breast cancer, and the HMLER cell line constructed from in vitro transformation of human mammary cells (Elenbaas et al, 2001; Morel et al, 2008). Data obtained indicated that the 5′-end CpGis of DAPK1 and NTN1 were methylated in the two cancer cell lines (Fig 2A). Furthermore, the treatment of MDA-MB-231 and HMLER cells with decitabine (DAC) resulted in a significant upregulation of DAPK1 and NTN1 mRNA (Fig 2B and C) and in the inhibition of DNA methylation within the DAPK1 and NTN1 promoter regions (Figs 2D and E, and EV1F and G). Parallel sequencing of DMRs (Fig 2A, dark gray boxes) validated their methylation status obtained from Methyl-Cap-seq experiments and indicated that DAC treatments reduced their level of methylation by half, in vitro (Fig 2D and E). Of note, DAC treatment of MDA-MB-231 cells resulted in the upregulation of UNC5B, although its promoter was not methylated in any of the cell lines tested (Fig 2B and C), suggesting an indirect regulatory mechanism. However, this upregulation was not observed for the other netrin-1-specific receptors, UNC5A, UNC5C, and DCC (Fig EV2A and B). Altogether, these data strongly suggest that DNA hypermethylation is involved in the transcriptional silencing of DAPK1 and NTN1 in human breast cancer cells. Click here to expand this figure. Figure EV2. The NTN1 DR apoptotic pathway, mechanism, and specificity A, B. Other NTN1 DR gene expression in breast cell lines, and impact of DAPK1 and NTN1 on the induction of apoptotic cell death in vitro. Gene expression was measured by qRT–PCR after 72 h in MDA-MB-231 (A) and HMLER (B) cells treated daily with 10 μM decitabine (DAC). The level of PBGD expression was used as an internal control. Data are represented as mean ± s.e.m. for to 3 independent experiments. ****P < 0.0001, two-tailed unpaired Student's t-test. C. Schematic representation of the cellular effect on HMLER cells of DAPK1 overexpression and/or recombinant netrin-1 treatment. D. Caspase-3 activation upon DAPK1 overexpression in HMLER and its reversion by the addition of netrin-1. ***P < 0.0001, one-way ANOVA. E. Schematic representation of DAC and effect of NTN1 siRNA on HMLER cells. F. Effect of NTN1 siRNA (siNTN1) on the expression of NTN1 by qRT–PCR. The level of PBGD expression was used as an internal control. ***P < 0.0001, one-way ANOVA. G, H. siNTN1 triggers apoptosis and cell death in hypomethylated HMLER cells. Cells were treated with DAC (10 μM, 72 h), and/or siNTN1 (30 pmol, 48 h), and caspase-3 activity (G) and cellular mortality (H) were measured. As a control, a scramble siRNA was used. Data (G, H) are expressed as mean ± s.e.m. of at least 3 independent experiments. ****P < 0.0001, one-way ANOVA. I, J. Representative images of TUNEL experiments shown in Fig 3, in MDA-MB-231 and HMLER cells, respectively. Scale bars = 50 μm. Download figure Download PowerPoint Decitabine resensitizes cancer cells to netrin-1 interference in vitro We thus hypothesized that the pharmacological targeting of the DNA methylation machinery may restore functional netrin-1/DR pathways and resensitize netrin-1-low cells to netrin-1 interference. We investigated whether the forced expression of DAPK1 in the DAPK1-negative HMLER cells re-established pro-apoptotic pathways (Fig EV2C and D). As expected, an increase in caspase-3 activity was observed in DAPK1 transfected cells. Furthermore, this pro-apoptotic effect was partially reversed by adding recombinant netrin-1 (Fig EV2D). We next analyzed cell death by conducting viability assays and caspase-3 activity assays in HMLER cells transfected with NTN1 siRNA and treated with DAC. While transfections had a mild effect on HMLER cells per se, treatment with DAC strongly potentiated the netrin-1 deprivation-induced cell death (Fig EV2F–H). Keeping in mind the therapeutic perspective of our study, we assessed the effect of combining DAC with a human anti-netrin-1 antibody, net1-mAb (Grandin et al, 2016). MDA-MB-231 and HMLER cells were treated with DAC or net1-mAb, or a combination of both drugs. As anticipated, the cell lines were resistant to net1-mAb alone, while, as previously observed (Lund et al, 2011) (Rodriguez-Paredes & Esteller, 2011b), DAC induced cell death in most of the cells tested, as evidenced by DNA fragmentation (Figs 3A and B, and EV2I and J) and viability assays (Fig 3C). The addition of net1-mAb to DAC-treated cells significantly enhanced apoptosis (Fig 3A–C). Moreover, this effect was blocked by the concomitant addition of recombinant netrin-1 (Fig 3A–C), indicating that net1-mAb-induced cell death was specifically linked to netrin-1 neutralization. To confirm that the pro-apoptotic activity observed upon the combined net1-mAb and DAC treatment was not linked to an intrinsic property of DAC, MDA-MB-231 and HMLER cell lines were treated with the 5-azacytidine (Aza), a DNA methylation inhibitor. This treatment resulted in similar gene expression modifications and cell death effects as DAC, when combined with the net1-mAb (Appendix Fig S1). The functional consequences of this combination treatment were also evaluated in additional human breast cancer cell lines. The highest upregulation of both NTN1 and DAPK1 was observed when treating cells with 10 μM of DAC (Fig EV3A–E), we thus used this concentration in the combined DAC and net1-mAb treatment to investigate the induction of apoptosis (Fig EV3F). A similar potentiation of cell death by combining DAC and net1-mAb was observed in cell lines (AU565, SKBR3, and MDA-MB-157) exhibiting an upregulation of at least one of 3 genes, NTN1, UNC5B, or DAPK1 (Fig EV3C–E). Figure 3. Netrin-1 neutralizing antibody net1-mAb triggers apoptosis
The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. The compound APR-246 (PRIMA-1Met/Eprenetapopt) is converted to methylene quinuclidinone (MQ) that targets mutant p53 protein and perturbs cellular antioxidant balance. APR-246 is currently tested in a phase III clinical trial in myelodysplastic syndrome (MDS). By in vitro, ex vivo, and in vivo models, we show that combined treatment with APR-246 and inhibitors of efflux pump MRP1/ABCC1 results in synergistic tumor cell death, which is more pronounced in TP53 mutant cells. This is associated with altered cellular thiol status and increased intracellular glutathione-conjugated MQ (GS-MQ). Due to the reversibility of MQ conjugation, GS-MQ forms an intracellular drug reservoir that increases availability of MQ for targeting mutant p53. Our study shows that redox homeostasis is a critical determinant of the response to mutant p53-targeted cancer therapy.
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