Faculty Bibliography

Breast cancer is a heterogeneous disease, and patients are categorized into subtypes according to gene expression. We studied the associations among molecular, immunohistochemical, and clinicopathologic features and their distribution according to the subtypes luminal, HER2, basal, and normal-like in 60 patients with invasive ductal breast carcinoma without distant metastasis at the time of diagnosis (M0). We evaluated the hypermethylation of the CDH-1, RASSF1A, SIAH-1 and TSLC-1 genes by methylation-specific polymerase chain reaction and the expression of p53, bcl-2, cyclin D1, E-cadherin, and beta-catenin proteins in tissue microarrays by immunohistochemical analysis. Expression of bcl-2 was associated with the luminal subtype (P=.003), and CDH-1 hypermethylation was present preferentially in HER2 tumors (P=.038). The basal subtype was characterized by the expression of beta-catenin (P=.003). The hypermethylation of CDH-1 and the expression of bcl-2, cyclin D1, and beta-catenin proteins differ among breast cancer subtypes.

Spry2 has been characterized as a negative regulator of the extracellular-regulated kinase (ERK) pathway. In this study we analysed whether epigenetic alterations of hSpry2 promoter occur in human lymphoid/hematopoietic malignancies. Our results revealed that hSpry2 promoter was hypermethylated in the HT cell line derived from a B-cell diffuse lymphoma, which correlated with decreased hSpry2 expression. We detected deregulation of the ERK pathway in these cells, but not in other blood cell lines expressing hSpry2. In addition, the ectopic overexpression of hSpry2 in HT cells drastically reduced the activation of ERK upon phorbol 12-myristate-13-acetate stimulation. Nude mice inoculated with HT mock cells developed tumors seven times larger than those from HT-hSpry2-transfected cells. We found hypermethylation of hSpry2 promoter in 37% (26 cases out of 71) of primary tumors from patients with B-cell diffuse lymphoma but none in normal B lymphocytes from 37 healthy individuals. Finally, we detected that hSpry2 promoter hypermethylation was associated with a significant decrease in the 5-year survival rate. These data suggest that hSpry2 could be important in lymphoid malignancies.

In order to determine new signal transduction pathways implicated in chronic myeloid leukaemia (CML), we performed a gene expression profile comparison between CD34+ cells from CML patients and healthy donors. Functional studies were performed using the Mo7e and Mo7e-p210 cell lines. Expression of CCND1 (Cyclin D1), as well as the chaperone HSPA8, which is important for regulation of CCND1, were significantly upregulated in CD34+ CML cells. Upregulation of HSPA8 was dependent, at least in part, on STAT5 (signal transducer and activator of transcrition 5)-dependent transcriptional activation, as demonstrated by chromatin immunoprecipitation. The presence of HSPA8 in the nuclear protein fraction as well as its binding to CCND1 suggests that it may contribute to stabilization of the CCND1/CDK4 complex, which, in turn, may participate in proliferation of CML cells. Treatment of CML cells with the specific HSPA8 inhibitor 15-deoxyspergualin induced inhibition of CML cell viability but did not induce apoptosis. In conclusion, our studies suggest that STAT5-mediated activation of HSPA8 induces nuclear translocation and activation of the CCND1/CDK4 complex leading to increased proliferation of CML cells, deciphering a new pathway implicated in CML and supporting a potential role of chaperone inhibitors in the treatment of CML.

Latent TGFbeta-binding protein 1 (LTBP-1) is a key regulator of TGFbeta targeting and activation in the extracellular matrix. LTBP-1 is recognized as a major docking molecule to localize, and possibly to activate, TGFbeta in the extracellular matrix. Despite this relevant function, the molecular mechanisms regulating Ltbp-1 transcription remain largely unknown. Previous results from our laboratory revealed that mouse embryonic fibroblasts (MEF) lacking dioxin receptor (AhR) had increased Ltbp-1 mRNA expression and elevated TGFbeta activity, suggesting that AhR repressed Ltbp-1 transcription. Here, we have cloned the mouse Ltbp-1 gene promoter and analysed its mechanism of transcriptional repression by AhR. Reporter gene assays, AhR over-expression and site-directed mutagenesis showed that basal Ltbp-1 transcription is AhR-dependent. Chromatin immunoprecipitation (ChIP) and RNA interference (RNAi) revealed that AhR regulates Ltbp-1 transcription by a mechanism involving recruitment of co-activators such as CREB1 and co-repressors such as HDAC2 to the Ltbp-1 promoter. In AhR-expressing (AhR+/+) MEF cells, the recruitment of HDAC1, 2 and 4 correlated with decreased K8H4 acetylation and impaired binding of pCREB(Ser133) to the Ltbp-1 promoter, likely maintaining a constitutive repressed state. AhR-/- MEF cells had the opposite pattern of HDACs and pCREB1(Ser133) binding to Ltbp-1 promoter, and therefore, over-expressed Ltbp-1 mRNA. In agreement, siRNA for HDAC2 increased Ltbp-1 expression and K8H4 acetylation in AhR+/+ but not in AhR-/- MEF cells. We suggest that HDAC2 binding keeps Ltbp-1 promoter repressed in AhR+/+ MEF cells, whereas in AhR-null MEF cells the absence of HDAC2 and the binding of pCREB(Ser133) allow Ltbp-1 transcription. Thus, epigenetics can contribute to constitutive Ltbp-1 repression by a mechanism requiring AhR activity.

Although disruption of histone modification patterns is a common hallmark of human cancer, our knowledge of the mechanistic role of histone-modifying enzymes in its generation is very limited. We have recently identified an inactivating mutation in the histone deacetylase-2 (HDAC2) in sporadic carcinomas with microsatellite instability and in tumors arising in individuals with hereditary nonpolyposis colorectal cancer syndrome. Since HDAC2 seems to be a central player in epigenetic gene repression, we wondered whether HDAC2-truncating mutations conferred a particular expression signature on these cancer cells. Using unsupervised clustering analysis in microsatellite-unstable colorectal cancer cell lines, we have found that HDAC2 mutant cells (RKO and Co115) show a characteristically different expression microarray signature from HDAC2 wild-type cells (HCT-116, SW48, HCT-15 and LoVo). HDAC2 mutant cells exhibit upregulation of tumor-promoting genes, such as those of tyrosine kinases, mediators of cell cycle progression and angiogenic factors. The overexpression of these genes is associated with a loss of HDAC2 recruitment and a gain of histone H4 hyperacetylation in their particular 5'-end promoters, as observed by chromatin immunoprecipitation. Transfection of wild-type HDAC2 in mutant cells reverted this epigenetic pattern by repressing the transforming genes in association with HDAC2 promoter occupancy. These results suggest a role for HDAC2 mutations in human tumorigenesis through the derepression of key genes from multiple cellular transformation pathways.

DNA methylation and the machinery involved in epigenetic regulation are key elements in the maintenance of cellular homeostasis. Epigenetic mechanisms are involved in embryonic development and the establishment of tissue-specific expression, X-chromosome inactivation and imprinting patterns, and maintenance of chromosome stability. The balance between all the enzymes and factors involved in DNA methylation and its interpretation by different groups of nuclear factors is crucial for normal cell behaviour. In cancer and other diseases, misregulation of epigenetic marks is a common feature, also including DNA methylation and histone post-translational modifications. In this scenario, it is worth mentioning a family of proteins characterized by the presence of a methyl-CpG-binding domain (MBDs) that are involved in interpreting the information encoded by DNA methylation and the recruitment of the enzymes responsible for establishing a silenced state of the chromatin. The generation of novel aberrantly hypermethylated regions during cancer development and progression makes MBD proteins interesting targets for their biological and clinical implications.

Methyl-cytosine-phosphate-guanine (CpG)-binding domain (MBD) proteins are bound to hypermethylated promoter CpG islands of tumor suppressor genes in human cancer cells, although a direct causal relationship at the genome-wide level between MBD presence and gene silencing remains to be demonstrated. To this end, we have inhibited the expression of MBD proteins in HeLa cells by short hairpin RNAs; and studied the functional consequences of MBD depletion using microarray-based expression analysis in conjunction with extensive bisulfite genomic sequencing and chromatin immunoprecipitation. The removal of MBDs results in a release of gene silencing associated with a loss of MBD occupancy in 5'-CpG islands without any change in the DNA methylation pattern. Our results unveil new targets for epigenetic inactivation mediated by MBDs in transformed cells, such as the cell adhesion protein gamma-parvin and the fibroblast growth factor 19, where we also demonstrate their bona fide tumor suppressor features. Our data support a fundamental role for MBD proteins in the direct maintenance of transcriptional repression of tumor suppressors and identify new candidate genes for epigenetic disruption in cancer cells.

An undifferentiated status and the epigenetic inactivation of tumor-suppressor genes are hallmarks of transformed cells. Promoter CpG island hypermethylation of differentiating genes, however, has rarely been reported. The Groucho homologue Transducin-like Enhancer of Split 1 (TLE1) is a multitasked transcriptional corepressor that acts through the acute myelogenous leukemia 1, Wnt, and Notch signaling pathways. We have found that TLE1 undergoes promoter CpG island hypermethylation-associated inactivation in hematologic malignancies, such as diffuse large B-cell lymphoma and AML. We also observed a mutual exclusivity of the epigenetic alteration of TLE1 and the cytogenetic alteration of AML1. TLE1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas TLE1-short hairpin RNA depletion in unmethylated cells enhances tumor growth. We also show that these effects are mediated by TLE1 transcriptional repressor activity on its target genes, such as Cyclin D1, Colony-Stimulating Factor 1 receptor, and Hairy/Enhancer of Split 1. These data suggest that TLE1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting critical differentiation and growth-suppressing pathways.

Changes in chromatin structure are an essential mechanism for gene regulation (transcription, replication, DNA repair and recombination). ATP-dependent chromatin-remodeling enzymes have previously been implicated in maintaining and regulating chromatin structure. These enzymes are divided into several groups including the SWI/SNF, ISWI and CHD families. Although the Chromodomain Helicase DNA-binding protein (CHD) family members are involved in key cellular processes, the disrupted regulation of their expression has not been fully assessed. An impairment of chromatin remodeling activity, mediated by promoter CpG island hypermethylation of the described candidate genes, could be key in cancer pathology. Herein, we examine the DNA methylation profiles of CHD family members (CHD1-9) in different tumor types. For all CHDs, CpG island hypermethylation was only observed at the CHD5 promoter in human cancer cell lines and primary tumors, particularly gliomas and colon and breast carcinomas. RT-qPCR analyses correlated CHD5 loss of expression with hypermethylation of the promoter, and restoration of CHD5 mRNA levels upon treatment with a DNA demethylating agent. These results underpin the epigenetic inactivation of the chromating remodeling factor CHD5 as one contributor for the aberrant structural changes of chromatin throughout the genome of the cancer cell.

BACKGROUND:DNA hypomethylation has been suggested to cause genomic instability and increase cancer risk. We aimed to test the hypothesis that DNA hypomethylation is associated with increased risk of bladder cancer. METHODS:We measured cytosine methylation (5-mC) content in genomic DNA from blood cells from patients with bladder cancer enrolled in a large case-control study in Spain between Jan 1, 1998, and Dec 31, 2001. Cases were men and women with newly diagnosed and histologically confirmed urothelial carcinoma of the bladder. Controls were selected from patients admitted to the same hospital for diseases or conditions unrelated to smoking or other known risk factors for bladder cancer. Controls were individually matched to cases on age (within 5 years), sex, race, and area of hospital referral. 5-mC content was measured in leucocyte DNA by use of a combination of high-performance capillary electrophoresis, Hpa II digestion, and densitometry. Data on demographics, 34 polymorphisms in nine folate metabolism genes, and nutritional intake of six B vitamins (including folate), alcohol, and smoking were assessed as potential confounders. Relative 5-mC content was expressed as a percentage (%5-mC) with respect to the total cytosine content (the sum of methylated and non-methylated cytosines). The primary endpoint was median %5-mC DNA content. FINDINGS/RESULTS:%5-mC was measured in leucocyte DNA from 775 cases and 397 controls. Median %5-mC DNA was significantly lower in cases (3.03% [IQR 2.17-3.56]) than in controls (3.19% [2.46-3.68], p=0.0002). All participants were subsequently categorised into quartiles by %5-mC content in controls. When the highest quartile of %5-mC content was used as the reference category (Q4), the following adjusted odds ratios (OR) and 95% CI were recorded for decreasing methylation quartiles: OR(Q3) 2.05 (95% CI 1.37-3.06); OR(Q2) 1.62 (1.07-2.44); and OR(Q1) 2.67 (1.77-4.03), p for trend <0.0001. The lowest cancer risk was noted in never smokers in the highest methylation quartile (never smokers in Q4). By comparison with never smokers in the highest quartile, current smokers in the lowest methylation quartile had the highest risk of bladder cancer (Q1: OR 25.51 [9.61-67.76], p for interaction 0.06). In analyses stratified by smoking, hypomethylation was a strong risk factor in never smokers (OR 6.39 [2.37-17.22]). Amount of methylation in controls were not associated with baseline characteristics, micronutrients, or selected genotypes in folate metabolism pathways. INTERPRETATION/CONCLUSIONS:For the first time, to our knowledge, we have shown in a large case-control study that leucocyte DNA hypomethylation is associated with increased risk of developing bladder cancer, and this association is independent of smoking and the other assessed risk factors. Amount of global methylation in genomic DNA could provide a useful biomarker of susceptibility to certain cancer types and further research is warranted.