Evidence of epigenetic changes affecting the chromatin state of the retinoic acid receptor beta2 promoter in breast cancer cells

Retinoic acid (RA)-resistance in breast cancer cells has been associated with irreversible loss of retinoic acid receptor beta, RARbeta, gene expression. Search of the causes affecting RARbeta gene activity has been oriented at identifying possible differences either at the level of one of the RARbeta promoters, RARbeta2, or at regulatory factors. We hypothesized that loss of RARbeta2 activity occurs as a result of multiple factors, including epigenetic modifications, which can pattern RARbeta2 chromatin state. Using methylation-specific PCR, we found hypermethylation at RARbeta2 in a significant proportion of both breast cancer cell lines and primary breast tumors. Treatment of cells with a methylated RARbeta2 promoter, by means of the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR), led to demethylation within RARbeta2 and expression of RARbeta indicating that DNA methylation is at least one factor, contributing to RARbeta inactivity. However, identically methylated promoters can differentially respond to RA, suggesting that RARbeta2 activity may be associated to different repressive chromatin states. This supposition is supported by the finding that the more stable repressive RARbeta2 state in the RA-resistant MDA-MB-231 cell line can be alleviated by the HDAC inhibitor, trichostatin A (TSA), with restoration of RA-induced RARbeta transcription. Thus, chromatin-remodeling drugs might provide a strategy to restore RARbeta activity, and help to overcome the hurdle of RA-resistance in breast cancer

A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples

The bisulfite genomic sequencing protocol is a widely used method for analyzing DNA methylation. It relies on the deamination of unmethylated cytosine residues to uracil; however, its high rates of DNA degradation and incomplete cytosine to uracil conversion often lead to failed experiments, uninformative results, and false positives. Here, we report the addition of a single-step multiple restriction enzyme digestion (MRED) designed to differentially digest polymerase chain reaction products amplified from unconverted DNA while leaving those of converted DNA intact. We show that for our model system, RARB2 P2 promoter, use of MRED increased informative sequencings ninefold, and MRED did not alter the clonal representation in one fully methylated cell line, H-596, treated or not with 5-azadeoxycytidine, a methylation inhibitor. We believe that this method may easily be adapted for analyzing other genes and provide guidelines for selecting the most appropriate MRED restriction enzymes

Misbehaviour of XIST RNA in Breast Cancer Cells

A role of X chromosome inactivation process in the development of breast cancer have been suggested. In particular, the relationship between the breast cancer predisposing gene BRCA1 and XIST, the main mediator of X chromosome inactivation, has been intensely investigated, but still remains controversial. We investigated this topic by assessing XIST behaviour in different groups of breast carcinomas and in a panel of breast cancer cell lines both BRCA1 mutant and wild type. In addition, we evaluated the occurrence of broader defects of heterochromatin in relation to BRCA1 status in breast cancer cells. We provide evidence that in breast cancer cells BRCA1 is involved in XIST regulation on the active X chromosome, but not in its localization as previously suggested, and that XIST can be unusually expressed by an active X and can decorate it. This indicates that the detection of XIST cloud in cancer cell is not synonymous of the presence of an inactive X chromosome. Moreover, we show that global heterochromatin defects observed in breast tumor cells are independent of BRCA1 status. Our observations sheds light on a possible previously uncharacterized mechanism of breast carcinogenesis mediated by XIST misbehaviour, particularly in BRCA1-related cancers. Moreover, the significant higher levels of XIST-RNA detected in BRCA1-associated respect to sporadic basal-like cancers, opens the possibility to use XIST expression as a marker to discriminate between the two groups of tumors

MGMT-Methylated Alleles Are Distributed Heterogeneously Within Glioma Samples Irrespective of IDH Status and Chromosome 10q Deletion

Several molecular markers drive diagnostic classification, prognostic stratification, and/or prediction of response to therapy in patients with gliomas. Among them, IDH gene mutations are valuable markers for defining subtypes and are strongly associated with epigenetic silencing of the methylguanine DNA methyltransferase (MGMT) gene. However, little is known about the percentage of MGMT-methylated alleles in IDH-mutated cells or the potential association between MGMT methylation and deletion of chromosome 10q, which encompasses the MGMT locus. Here, we quantitatively assessed MGMT methylation and IDH1 mutation in 208 primary glioma samples to explore possible differences associated with the IDH genotype. We also explored a potential association between MGMT methylation and loss of chromosome 10q. We observed that MGMT methylation was heterogeneously distributed within glioma samples irrespective of IDH status suggesting an incomplete overlap between IDH1-mutated and MGMT-methylated alleles and indicating a partial association between these two events. Moreover, loss of one MGMT allele did not affect the methylation level of the remaining allele. MGMT was methylated in about half of gliomas harboring a 10q deletion; in those cases, loss of heterozygosity might be considered a second hit leading to complete inactivation of MGMT and further contributing to tumor progression

Derangement of a Factor Upstream of RARα Triggers the Repression of a Pleiotropic Epigenetic Network

Chromatin adapts and responds to extrinsic and intrinsic cues. We hypothesize that inheritable aberrant chromatin states in cancer and aging are caused by genetic/environmental factors. In previous studies we demonstrated that either genetic mutations, or loss, of retinoic acid receptor alpha (RARalpha), can impair the integration of the retinoic acid (RA) signal at the chromatin of RA-responsive genes downstream of RARalpha, and can lead to aberrant repressive chromatin states marked by epigenetic modifications. In this study we tested whether the mere interference with the availability of RA signal at RARalpha, in cells with an otherwise functional RARalpha, can also induce epigenetic repression at RA-responsive genes downstream of RARalpha.To hamper the availability of RA at RARalpha in untransformed human mammary epithelial cells, we targeted the cellular RA-binding protein 2 (CRABP2), which transports RA from the cytoplasm onto the nuclear RARs. Stable ectopic expression of a CRABP2 mutant unable to enter the nucleus, as well as stable knock down of endogenous CRABP2, led to the coordinated transcriptional repression of a few RA-responsive genes downstream of RARalpha. The chromatin at these genes acquired an exacerbated repressed state, or state "of no return". This aberrant state is unresponsive to RA, and therefore differs from the physiologically repressed, yet "poised" state, which is responsive to RA. Consistent with development of homozygosis for epigenetically repressed loci, a significant proportion of cells with a defective CRABP2-mediated RA transport developed heritable phenotypes indicative of loss of function.Derangement/lack of a critical factor necessary for RARalpha function induces epigenetic repression of a RA-regulated gene network downstream of RARalpha, with major pleiotropic biological outcomes

SETBP1 variants outside the degron disrupt DNA-binding and transcription independent of protein abundance to cause a heterogeneous neurodevelopmental disorder

Germline de novo SETBP1 variants cause clinically distinct and heterogeneous neurodevelopmental disorders. Heterozygous missense variants at a hotspot encoding a canonical degron lead to SETBP1 accumulation and Schinzel-Giedion syndrome (SGS), a rare severe developmental disorder involving multisystem malformations. Heterozygous loss-of-function variants result in SETBP1 haploinsufficiency disorder which is phenotypically much milder than SGS. Following an initial description of four individuals with atypical SGS carrying heterozygous missense variants adjacent to the degron, a few individual cases of variants outside the degron were reported. Due to the lack of systematic investigation of genotype-phenotype associations of different types of SETBP1 variants, and limited understanding of the roles of the gene in brain development, the extent of clinical heterogeneity and how this relates to underlying pathophysiological mechanisms remain elusive, imposing challenges for diagnosis and patient care. Here, we present a comprehensive investigation of the largest cohort to-date of individuals carrying SETBP1 missense variants outside the degron (n=18, including one in-frame deletion). We performed thorough clinical and speech phenotyping with functional follow-up using cellular assays and transcriptomics. Our findings suggest that such variants cause a clinically and functionally variable developmental syndrome, showing only partial overlaps with classical SGS and SETBP1 haploinsufficiency disorder, and primarily characterised by intellectual disability, epilepsy, speech and motor impairment. We provide evidence of loss-of-function pathophysiological mechanisms impairing ubiquitination, DNA-binding and transcription. In contrast to SGS and SETBP1 haploinsufficiency, these effects are independent of protein abundance. Overall, our study provides important novel insights into diagnosis, patient care and aetiology of SETBP1-related disorders

Constitutive BRCA1 Promoter Hypermethylation Can Be a Predisposing Event in Isolated Early-Onset Breast Cancer

Early age at onset of breast cancer (eoBC) is suggestive of an increased genetic risk. Although genetic testing is offered to all eoBC-affected women, in isolated cases the detection rate of pathogenic variants is 60% and loss of heterozygosity at chromosome 17q. The patient hypermethylated at RAD51C showed low methylation in the tumor sample, ruling out a role for methylation-induced silencing in tumor development. In isolated eoBC patients, BRCA1 constitutive promoter methylation may be a predisposing event. Further studies are required to define the impact of methylation changes occurring at BC-predisposing genes and their role in tumorigenesis

Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells

BACKGROUND: The retinoic acid receptor beta 2 (RARβ2) gene modulates proliferation and survival of cultured human breast cancer cells. Previously we showed that ectopic expression of RARβ2 in a mouse xenograft model prevented metastasis, even in the absence of the ligand, all-trans retinoic acid. We investigated both cultured cells and xenograft tumors in order to delineate the gene expression profiles responsible for an antimetastatic phenotype. METHODS: RNA from MDA-MB-435 human breast cancer cells transduced with RARβ2 or empty retroviral vector (LXSN) was analyzed using Agilent Human 1A Oligo microarrays. The one hundred probes with the greatest differential intensity (p < 0.004, jointly) were determined by selecting the top median log ratios from eight-paired microarrays. Validation of differences in expression was done using Northern blot analysis and quantitative RT-PCR (qRT-PCR). We determined expression of selected genes in xenograft tumors. RESULTS: RARβ2 cells exhibit gene profiles with overrepresentation of genes from Xq28 (p = 2 × 10(-8)), a cytogenetic region that contains a large portion of the cancer/testis antigen gene family. Other functions or factors impacted by the presence of exogenous RARβ2 include mediators of the immune response and transcriptional regulatory mechanisms. Thirteen of fifteen (87%) of the genes evaluated in xenograft tumors were consistent with differences we found in the cell cultures (p = 0.007). CONCLUSION: Antimetastatic RARβ2 signalling, direct or indirect, results in an elevation of expression for genes such as tumor-cell antigens (CTAG1 and CTAG2), those involved in innate immune response (e.g., RIG-I/DDX58), and tumor suppressor functions (e.g., TYRP1). Genes whose expression is diminished by RARβ2 signalling include cell adhesion functions (e.g, CD164) nutritional or metabolic processes (e.g., FABP6), and the transcription factor, JUN

Variant-specific pathophysiological mechanisms of AFF3 differently influence transcriptome profiles

Background We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney, caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative mode of action, wherein an increased level of AFF3 resulted in pathological effects. Methods Evolutionary constraints suggest that other modes-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be damaging variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants. Results We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous Loss-of-Function (LoF) or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not rescue these phenotypes. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring + / + , KINSSHIP/KINSSHIP, LoF/ + , LoF/LoF or KINSSHIP/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the KINSSHIP/KINSSHIP or the LoF/LoF lines. While the same pathways are affected, only about one third of the differentially expressed genes are common to the homozygote datasets, indicating that AFF3 LoF and KINSSHIP variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation. Conclusions Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious

Cigarette smoke exposure inhibits extracellular MMP-2 (gelatinase A) activity in human lung fibroblasts

BACKGROUND: Exposure to cigarette smoke is considered a major risk factor for the development of lung diseases, since its causative role has been assessed in the induction and maintenance of an inflamed state in the airways. Lung fibroblasts can contribute to these processes, due to their ability to produce proinflammatory chemotactic molecules and extracellular matrix remodelling proteinases. Among proteolytic enzymes, gelatinases A and B have been studied for their role in tissue breakdown and mobilisation of matrix-derived signalling molecules. Multiple reports linked gelatinase deregulation and overexpression to the development of inflammatory chronic lung diseases such as COPD. METHODS: In this study we aimed to determine variations in the gelatinolytic pattern of human lung fibroblasts (HFL-1 cell line) exposed to cigarette smoke extract (CSE). Gelatinolytic activity levels were determined by using gelatin zymography for the in-gel detection of the enzymes (proenzyme and activated forms), and the subsequent semi-quantitative densitometric evaluation of lytic bands. Expression of gelatinases was evaluated also by RT-PCR, zymography of the cell lysates and by western blotting. RESULTS: CSE exposure at the doses used (1–10%) did not exert any significant cytotoxic effects on fibroblasts. Zymographic analysis showed that CSE exposure resulted in a linear decrease of the activity of gelatinase A. Control experiments allowed excluding a direct inhibitory effect of CSE on gelatinases. Zymography of cell lysates confirmed the expression of MMP-2 in all conditions. Semi-quantitative evaluation of mRNA expression allowed assessing a reduced transcription of the enzyme, as well as an increase in the expression of TIMP-2. Statistical analyses showed that the decrease of MMP-2 activity in conditioned media reached the statistical significance (p = 0.0031 for 24 h and p = 0.0012 for 48 h), while correlation analysis showed that this result was independent from CSE cytotoxicity (p = 0.7833 for both exposures). CONCLUSION: Present work describes for the first time that, apart well characterized proinflammatory responses, human lung fibroblasts may react to CSE with a significant reduction of extracellular MMP-2 lytic activity. Therefore, fibroblasts may actively participate to the alteration of the proteolysis/antiproteolysis balance, which reflects the defective repair of the extracellular matrix. Such event should provide a further contribution to the maintenance of the inflamed state in the lungs