ABSTRACT
The experimental need to engineer the genome both in time and space, has led to the development of several photoactivatable Cre recombinase systems. However, the combination of inefficient and non-intentional background recombination has prevented thus far the wide application of these systems in biological and biomedical research. Here, we engineer an optimized photoactivatable Cre recombinase system that we refer to as doxycycline- and light-inducible Cre recombinase (DiLiCre). Following extensive characterization in cancer cell and organoid systems, we generate a DiLiCre mouse line, and illustrated the biological applicability of DiLiCre for light-induced mutagenesis in vivo and positional cell-tracing by intravital microscopy. These experiments illustrate how newly formed HrasV12 mutant cells follow an unnatural movement towards the interfollicular dermis. Together, we develop an efficient photoactivatable Cre recombinase mouse model and illustrate how this model is a powerful genome-editing tool for biological and biomedical research.
Subject(s)
Doxycycline , Optogenetics , Mice , Animals , Doxycycline/pharmacology , Mice, Transgenic , Gene Editing , Integrases/genetics , Integrases/metabolism , Mice, Inbred StrainsABSTRACT
The morphology and functionality of the epithelial lining differ along the intestinal tract, but tissue renewal at all sites is driven by stem cells at the base of crypts1-3. Whether stem cell numbers and behaviour vary at different sites is unknown. Here we show using intravital microscopy that, despite similarities in the number and distribution of proliferative cells with an Lgr5 signature in mice, small intestinal crypts contain twice as many effective stem cells as large intestinal crypts. We find that, although passively displaced by a conveyor-belt-like upward movement, small intestinal cells positioned away from the crypt base can function as long-term effective stem cells owing to Wnt-dependent retrograde cellular movement. By contrast, the near absence of retrograde movement in the large intestine restricts cell repositioning, leading to a reduction in effective stem cell number. Moreover, after suppression of the retrograde movement in the small intestine, the number of effective stem cells is reduced, and the rate of monoclonal conversion of crypts is accelerated. Together, these results show that the number of effective stem cells is determined by active retrograde movement, revealing a new channel of stem cell regulation that can be experimentally and pharmacologically manipulated.
Subject(s)
Cell Count , Cell Movement , Intestines , Stem Cells , Animals , Intestinal Mucosa/cytology , Intestine, Small/cytology , Intestines/cytology , Mice , Receptors, G-Protein-Coupled , Stem Cells/cytology , Wnt ProteinsABSTRACT
(1) Background: an increasing number of breast cancer patients develop lethal brain metastases (BM). The complete removal of these tumors by surgery becomes complicated when cells infiltrate into the brain parenchyma. However, little is known about the nature of these invading cells in breast cancer brain metastasis (BCBM). (2) Methods: we use intravital microscopy through a cranial window to study the behavior of invading cells in a mouse model of BCBM. (3) Results: we demonstrate that BCBM cells that escape from the metastatic mass and infiltrate into brain parenchyma undergo epithelial-to-mesenchymal transition (EMT). Moreover, cells undergoing EMT revert to an epithelial state when growing tumor masses in the brain. Lastly, through multiplex immunohistochemistry, we confirm the presence of these infiltrative cells in EMT in patient samples. (4) Conclusions: together, our data identify the critical role of EMT in the invasive behavior of BCBM, which warrants further consideration to target those cells when treating BCBM.
ABSTRACT
DNA methylation is an epigenetic process that controls DNA accessibility and serves as a transcriptomic switch when deposited at regulatory regions. The adequate functioning of this process is indispensable for tissue homeostasis and cell fate determination. Conversely, altered DNA methylation patterns result in abnormal gene transcription profiles that contribute to tumor initiation and progression. However, whether the consequence of DNA methylation on gene expression and cell fate is uniform regardless of the cell type or state could so far not been tested due to the lack of technologies to target DNA methylation in-situ. Here, we have taken advantage of CRISPR/dCas9 technology adapted for epigenetic editing through site-specific targeting of DNA methylation to characterize the transcriptional changes of the candidate gene and the functional effects on cell fate in different tumor settings. As a proof-of-concept, we were able to induce de-novo site-specific methylation of the gene promoter of IGFBP2 up to 90% with long-term and bona-fide inheritance by daughter cells. Strikingly, this modification led to opposing expression profiles of the target gene in different cancer cell models and affected the expression of mesenchymal genes CDH1, VIM1, TGFB1 and apoptotic marker BCL2. Moreover, methylation-induced changes in expression profiles was also accompanied by a phenotypic switch in cell migration and cell morphology. We conclude that in different cell types the consequence of DNA methylation on gene expression and cell fate can be completely different.
ABSTRACT
Lung cancer is the leading cause of cancer-related death worldwide. The desmoplastic stroma of lung cancer and other solid tumors is rich in tumor-associated fibroblasts (TAFs) exhibiting an activated/myofibroblast-like phenotype. There is growing awareness that TAFs support key steps of tumor progression and are epigenetically reprogrammed compared to healthy fibroblasts. Although the mechanisms underlying such epigenetic reprogramming are incompletely understood, there is increasing evidence that they involve interactions with either cancer cells, pro-fibrotic cytokines such as TGF-ß, the stiffening of the surrounding extracellular matrix, smoking cigarette particles and other environmental cues. These aberrant interactions elicit a global DNA hypomethylation and a selective transcriptional repression through hypermethylation of the TGF-ß transcription factor SMAD3 in lung TAFs. Likewise, similar DNA methylation changes have been reported in TAFs from other cancer types, as well as histone core modifications and altered microRNA expression. In this review we summarize the evidence of the epigenetic reprogramming of TAFs, how this reprogramming contributes to the acquisition and maintenance of a tumor-promoting phenotype, and how it provides novel venues for therapeutic intervention, with a special focus on lung TAFs.
ABSTRACT
The tumor-promoting fibrotic stroma rich in tumor-associated fibroblasts (TAF) is drawing increased therapeutic attention. Intriguingly, a trial with the antifibrotic drug nintedanib in non-small cell lung cancer reported clinical benefits in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC), even though the stroma is fibrotic in both histotypes. Likewise, we reported that nintedanib inhibited the tumor-promoting fibrotic phenotype of TAFs selectively in ADC. Here we show that tumor fibrosis is actually higher in ADC-TAFs than SCC-TAFs in vitro and patient samples. Mechanistically, the reduced fibrosis and nintedanib response of SCC-TAFs was associated with increased promoter methylation of the profibrotic TGFß transcription factor SMAD3 compared with ADC-TAFs, which elicited a compensatory increase in TGFß1/SMAD2 activation. Consistently, forcing global DNA demethylation of SCC-TAFs with 5-AZA rescued TGFß1/SMAD3 activation, whereas genetic downregulation of SMAD3 in ADC-TAFs and control fibroblasts increased TGFß1/SMAD2 activation, and reduced their fibrotic phenotype and antitumor responses to nintedanib in vitro and in vivo. Our results also support that smoking and/or the anatomic location of SCC in the proximal airways, which are more exposed to cigarette smoke particles, may prime SCC-TAFs to stronger SMAD3 epigenetic repression, because cigarette smoke condensate selectively increased SMAD3 promoter methylation. Our results unveil that the histotype-specific regulation of tumor fibrosis in lung cancer is mediated through differential SMAD3 promoter methylation in TAFs and provide new mechanistic insights on the selective poor response of SCC-TAFs to nintedanib. Moreover, our findings support that patients with ADC may be more responsive to antifibrotic drugs targeting their stromal TGFß1/SMAD3 activation. SIGNIFICANCE: This study implicates the selective epigenetic repression of SMAD3 in SCC-TAFs in the clinical failure of nintedanib in SCC and supports that patients with ADC may benefit from antifibrotic drugs targeting stromal TGFß1/SMAD3.
Subject(s)
Adenocarcinoma of Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Resistance, Neoplasm/genetics , Indoles/pharmacology , Lung Neoplasms/drug therapy , Smad3 Protein/genetics , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/pathology , Adenocarcinoma of Lung/surgery , Aged , Aged, 80 and over , Animals , Cancer-Associated Fibroblasts/drug effects , Cancer-Associated Fibroblasts/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/surgery , Cohort Studies , DNA Methylation/genetics , Epigenetic Repression , Female , Fibrosis , Gene Expression Regulation, Neoplastic , Humans , Indoles/therapeutic use , Lung/cytology , Lung/drug effects , Lung/pathology , Lung/surgery , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/surgery , Male , Mice , Middle Aged , Pneumonectomy , Promoter Regions, Genetic/genetics , Smad2 Protein/genetics , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Tissue Array Analysis , Xenograft Model Antitumor AssaysABSTRACT
HP1 is a structural component of heterochromatin. Mammalian HP1 isoforms HP1α, HP1ß, and HP1γ play different roles in genome stability, but their precise role in heterochromatin structure is unclear. Analysis of Hp1α-/-, Hp1ß-/-, and Hp1γ-/- MEFs show that HP1 proteins have both redundant and unique functions within pericentric heterochromatin (PCH) and also act globally throughout the genome. HP1α confines H4K20me3 and H3K27me3 to regions within PCH, while its absence results in a global hyper-compaction of chromatin associated with a specific pattern of mitotic defects. In contrast, HP1ß is functionally associated with Suv4-20h2 and H4K20me3, and its loss induces global chromatin decompaction and an abnormal enrichment of CTCF in PCH and other genomic regions. Our work provides insight into the roles of HP1 proteins in heterochromatin structure and genome stability.
Subject(s)
Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Amino Acid Sequence/genetics , Animals , Chromatin/metabolism , Chromobox Protein Homolog 5 , HeLa Cells , Humans , Mammals/metabolism , Protein Binding/genetics , Protein Binding/immunology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
BACKGROUND: Cutaneous melanoma is the deadliest skin cancer, with an increasing incidence and mortality rate. Currently, staging of patients with primary melanoma is performed using histological biomarkers such as tumor thickness and ulceration. As disruption of the epigenomic landscape is recognized as a widespread feature inherent in tumor development and progression, we aimed to identify novel biomarkers providing additional clinical information over current factors using unbiased genome-wide DNA methylation analyses. METHODS: We performed a comprehensive DNA methylation analysis during all progression stages of melanoma using Infinium HumanMethylation450 BeadChips on a discovery cohort of benign nevi (n = 14) and malignant melanoma from both primary (n = 33) and metastatic (n = 28) sites, integrating the DNA methylome with gene expression data. We validated the discovered biomarkers in three independent validation cohorts by pyrosequencing and immunohistochemistry. RESULTS: We identified and validated biomarkers for, and pathways involved in, melanoma development (e.g., HOXA9 DNA methylation) and tumor progression (e.g., TBC1D16 DNA methylation). In addition, we determined a prognostic signature with potential clinical applicability and validated PON3 DNA methylation and OVOL1 protein expression as biomarkers with prognostic information independent of tumor thickness and ulceration. CONCLUSIONS: Our data underscores the importance of epigenomic regulation in triggering metastatic dissemination through the inactivation of central cancer-related pathways. Inactivation of cell-adhesion and differentiation unleashes dissemination, and subsequent activation of inflammatory and immune system programs impairs anti-tumoral defense pathways. Moreover, we identify several markers of tumor development and progression previously unrelated to melanoma, and determined a prognostic signature with potential clinical utility.
Subject(s)
DNA Methylation , DNA, Neoplasm/metabolism , Melanoma/genetics , Melanoma/physiopathology , Skin Neoplasms/genetics , Skin Neoplasms/physiopathology , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Disease Progression , Female , Humans , Male , Middle Aged , Prognosis , Melanoma, Cutaneous MalignantABSTRACT
BACKGROUND: Cancer patients often show no or only modest benefit from a given therapy. This major problem in oncology is generally attributed to the lack of specific predictive biomarkers, yet a global measure of cancer cell activity may support a comprehensive mechanistic understanding of therapy efficacy. We reasoned that network analysis of omic data could help to achieve this goal. METHODS: A measure of "cancer network activity" (CNA) was implemented based on a previously defined network feature of communicability. The network nodes and edges corresponded to human proteins and experimentally identified interactions, respectively. The edges were weighted proportionally to the expression of the genes encoding for the corresponding proteins and relative to the number of direct interactors. The gene expression data corresponded to the basal conditions of 595 human cancer cell lines. Therapeutic responses corresponded to the impairment of cell viability measured by the half maximal inhibitory concentration (IC50) of 130 drugs approved or under clinical development. Gene ontology, signaling pathway, and transcription factor-binding annotations were taken from public repositories. Predicted synergies were assessed by determining the viability of four breast cancer cell lines and by applying two different analytical methods. RESULTS: The effects of drug classes were associated with CNAs formed by different cell lines. CNAs also differentiate target families and effector pathways. Proteins that occupy a central position in the network largely contribute to CNA. Known key cancer-associated biological processes, signaling pathways, and master regulators also contribute to CNA. Moreover, the major cancer drivers frequently mediate CNA and therapeutic differences. Cell-based assays centered on these differences and using uncorrelated drug effects reveals novel synergistic combinations for the treatment of breast cancer dependent on PI3K-mTOR signaling. CONCLUSIONS: Cancer therapeutic responses can be predicted on the basis of a systems-level analysis of molecular interactions and gene expression. Fundamental cancer processes, pathways, and drivers contribute to this feature, which can also be exploited to predict precise synergistic drug combinations.
Subject(s)
Antineoplastic Agents/pharmacology , Drugs, Investigational/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Gene Regulatory Networks/drug effects , Neoplasm Proteins/genetics , Prescription Drugs/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Survival/drug effects , Drug Synergism , Female , Gene Expression Profiling , Gene Ontology , Humans , Molecular Sequence Annotation , Mutation , Neoplasm Proteins/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolismABSTRACT
BACKGROUND: One of the hallmarks of cancer is the disruption of gene expression patterns. Many molecular lesions contribute to this phenotype, and the importance of aberrant DNA methylation profiles is increasingly recognized. Much of the research effort in this area has examined proximal promoter regions and epigenetic alterations at other loci are not well characterized. RESULTS: Using whole genome bisulfite sequencing to examine uncharted regions of the epigenome, we identify a type of far-reaching DNA methylation alteration in cancer cells of the distal regulatory sequences described as super-enhancers. Human tumors undergo a shift in super-enhancer DNA methylation profiles that is associated with the transcriptional silencing or the overactivation of the corresponding target genes. Intriguingly, we observe locally active fractions of super-enhancers detectable through hypomethylated regions that suggest spatial variability within the large enhancer clusters. Functionally, the DNA methylomes obtained suggest that transcription factors contribute to this local activity of super-enhancers and that trans-acting factors modulate DNA methylation profiles with impact on transforming processes during carcinogenesis. CONCLUSIONS: We develop an extensive catalogue of human DNA methylomes at base resolution to better understand the regulatory functions of DNA methylation beyond those of proximal promoter gene regions. CpG methylation status in normal cells points to locally active regulatory sites at super-enhancers, which are targeted by specific aberrant DNA methylation events in cancer, with putative effects on the expression of downstream genes.
Subject(s)
DNA Methylation/genetics , Epigenomics , Neoplasms/genetics , CpG Islands/genetics , Genome, Human , High-Throughput Nucleotide Sequencing , Humans , Promoter Regions, GeneticSubject(s)
Antineoplastic Agents/therapeutic use , Melanoma/drug therapy , Molecular Targeted Therapy , Protein Kinase Inhibitors/therapeutic use , Skin Neoplasms/drug therapy , DNA Methylation/drug effects , Epigenesis, Genetic , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , Humans , Melanoma/genetics , Melanoma/pathology , Promoter Regions, Genetic , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Recurrence , Skin Neoplasms/genetics , Skin Neoplasms/pathologyABSTRACT
The discovery of highly recurrent mutations in melanoma, such as BRAF(V600E), completely changed the clinical management including therapy of melanoma patients. In the era of Personalized Medicine targeted melanoma therapies showed high response rates, currently evidenced by BRAF inhibitors or immune-stimulating therapies. In addition to genetic biomarkers, epigenetic knowledge in melanoma has undergone a major step forward in recent years. In particular, epigenetics is unveiling new perspectives to fight this disease, providing an encouraging number of DNA methylation based biomarkers that will likely improve patient stratification for prognosis and treatment. In this regard, putative targetable biomarkers or those with predictive value for the outcome of currently applied therapies are promising tools for future precision oncology strategies. In addition, the progress made in genetic and epigenetic profiling technologies and their reconfiguration to real-time clinical screening approaches makes personalized medicine in melanoma an achievable objective in upcoming years.
ABSTRACT
Epigenetic changes through altered DNA methylation have been implicated in critical aspects of tumor progression, and have been extensively studied in a variety of cancer types. In contrast, our current knowledge of the aberrant genomic DNA methylation in tumor-associated fibroblasts (TAFs) or other stromal cells that act as critical coconspirators of tumor progression is very scarce. To address this gap of knowledge, we conducted genome-wide DNA methylation profiling on lung TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer patients using the HumanMethylation450 microarray. We found widespread DNA hypomethylation concomitant with focal gain of DNA methylation in TAFs compared to CFs. The aberrant DNA methylation landscape of TAFs had a global impact on gene expression and a selective impact on the TGF-ß pathway. The latter included promoter hypermethylation-associated SMAD3 silencing, which was associated with hyperresponsiveness to exogenous TGF-ß1 in terms of contractility and extracellular matrix deposition. In turn, activation of CFs with exogenous TGF-ß1 partially mimicked the epigenetic alterations observed in TAFs, suggesting that TGF-ß1 may be necessary but not sufficient to elicit such alterations. Moreover, integrated pathway-enrichment analyses of the DNA methylation alterations revealed that a fraction of TAFs may be bone marrow-derived fibrocytes. Finally, survival analyses using DNA methylation and gene expression datasets identified aberrant DNA methylation on the EDARADD promoter sequence as a prognostic factor in non-small cell lung cancer patients. Our findings shed light on the unique origin and molecular alterations underlying the aberrant phenotype of lung TAFs, and identify a stromal biomarker with potential clinical relevance.
Subject(s)
Adenocarcinoma/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Squamous Cell/genetics , DNA Methylation , Fibroblasts/metabolism , Lung Neoplasms/genetics , Adenocarcinoma/pathology , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Squamous Cell/pathology , Epigenesis, Genetic , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Focal Adhesions/genetics , Focal Adhesions/metabolism , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/pathology , Male , MicroRNAs/genetics , MicroRNAs/metabolism , Middle Aged , Receptors, IgG/genetics , Receptors, IgG/metabolism , Sequence Analysis, DNA , Smad3 Protein/genetics , Smad3 Protein/metabolism , Transcription, Genetic , Transforming Growth Factor beta1/physiology , Tumor Cells, CulturedABSTRACT
Metastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors.
Subject(s)
Disease Progression , Epigenesis, Genetic , ErbB Receptors/metabolism , GTPase-Activating Proteins/genetics , Melanoma/genetics , Melanoma/pathology , Animals , Cell Line, Tumor , DNA Methylation/drug effects , DNA Methylation/genetics , Epigenesis, Genetic/drug effects , GTPase-Activating Proteins/metabolism , Immunoprecipitation , Mice, Nude , Molecular Weight , Neoplasm Metastasis , Prognosis , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinase Inhibitors/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Signal Transduction/drug effects , Transcriptional Activation/drug effects , Transcriptional Activation/genetics , Treatment Outcome , rab GTP-Binding Proteins/metabolismABSTRACT
Cancer evolution is a process that is still poorly understood because of the lack of versatile in vivo longitudinal studies. By generating murine non-small cell lung cancer (NSCLC) orthoallobanks and paired primary cell lines, we provide a detailed description of an in vivo, time-dependent cancer malignization process. We identify the acquisition of metastatic dissemination potential, the selection of co-driver mutations, and the appearance of naturally occurring intratumor heterogeneity, thus recapitulating the stochastic nature of human cancer development. This approach combines the robustness of genetically engineered cancer models with the flexibility of allograft methodology. We have applied this tool for the preclinical evaluation of therapeutic approaches. This system can be implemented to improve the design of future treatments for patients with NSCLC.
Subject(s)
Biological Evolution , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/genetics , Neoplasms, Experimental/genetics , Allografts/drug effects , Allografts/pathology , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Disease Models, Animal , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Mice , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathologyABSTRACT
Epithelial-to-mesenchymal transition (EMT) is a plastic process in which fully differentiated epithelial cells are converted into poorly differentiated, migratory and invasive mesenchymal cells, and it has been related to the metastasis potential of tumors. This is a reversible process and cells can also eventually undergo mesenchymal-to-epithelial transition. The existence of a dynamic EMT process suggests the involvement of epigenetic shifts in the phenotype. Herein, we obtained the DNA methylomes at single-base resolution of Madin-Darby canine kidney cells undergoing EMT and translated the identified differentially methylated regions to human breast cancer cells undergoing a gain of migratory and invasive capabilities associated with the EMT phenotype. We noticed dynamic and reversible changes of DNA methylation, both on promoter sequences and gene-bodies in association with transcription regulation of EMT-related genes. Most importantly, the identified DNA methylation markers of EMT were present in primary mammary tumors in association with the epithelial or the mesenchymal phenotype of the studied breast cancer samples.
Subject(s)
DNA Methylation , Epithelial-Mesenchymal Transition , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Dogs , Female , Humans , Madin Darby Canine Kidney CellsABSTRACT
INTRODUCTION: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process. METHODS: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. RESULTS: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. CONCLUSIONS: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.
Subject(s)
Breast Neoplasms/drug therapy , Drug Resistance, Neoplasm/genetics , Estrogen Receptor alpha/metabolism , Indazoles/pharmacology , Proto-Oncogene Proteins c-vav/genetics , Androstadienes/therapeutic use , Antineoplastic Agents, Hormonal/pharmacology , Aromatase Inhibitors/therapeutic use , Biomarkers, Tumor/genetics , Breast/pathology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Enzyme Activators/pharmacology , ErbB Receptors/antagonists & inhibitors , Erlotinib Hydrochloride , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/genetics , Female , Gene Expression Regulation, Neoplastic , Genetic Association Studies , Genetic Variation , Humans , Letrozole , MCF-7 Cells , Nitriles/therapeutic use , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , RNA Interference , RNA, Small Interfering , Tamoxifen/pharmacology , Tamoxifen/therapeutic use , Toremifene/pharmacology , Toremifene/therapeutic use , Triazoles/therapeutic useABSTRACT
A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.
Subject(s)
DNA Methylation , DNA Repair , Oligonucleotide Array Sequence Analysis/methods , Fixatives , Formaldehyde , Humans , Paraffin EmbeddingABSTRACT
Cancer is as much an epigenetic disease as a genetic one; however, the interplay between these two processes is unclear. Recently, it has been shown that a large proportion of DNA methylation variability can be explained by allele-specific methylation (ASM), either at classical imprinted loci or those regulated by underlying genetic variants. During a recent screen for imprinted differentially methylated regions, we identified the genomic interval overlapping the non-coding nc886 RNA (previously known as vtRNA2-1) as an atypical ASM that shows variable levels of methylation, predominantly on the maternal allele in many tissues. Here we show that the nc886 interval is the first example of a polymorphic imprinted DMR in humans. Further analysis of the region suggests that the interval subjected to ASM is approximately 2 kb in size and somatically acquired. An in depth analysis of this region in primary cancer samples with matching normal adjacent tissue from the Cancer Genome Atlas revealed that aberrant methylation in bladder, breast, colon and lung tumors occurred in approximately 27% of cases. Hypermethylation occurred more frequently than hypomethylation. Using additional normal-tumor paired samples we show that on rare occasions the aberrant methylation profile is due to loss-of-heterozygosity. This work therefore suggests that the nc886 locus is subject to variable allelic methylation that undergoes cancer-associated epigenetic changes in solid tumors.