Hector Hernandez-Vargas

Hepatocellular Carcinoma Displays Distinct DNA Methylation Signatures with Potential as Clinical Predictors

Background Hepatocellular carcinoma (HCC) is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be the cause of its molecular and clinicopathological heterogeneity. A better understanding of the global deregulation of methylation states and how they correlate with disease progression will aid in the design of strategies for earlier detection and better therapeutic decisions. Methods and Findings We characterized the changes in promoter methylation in a series of 30 HCC tumors and their respective surrounding tissue and identified methylation signatures associated with major risk factors and clinical correlates. A wide panel of cancer-related gene promoters was analyzed using Illumina bead array technology, and CpG sites were then selected according to their ability to classify clinicopathological parameters. An independent series of HCC tumors and matched surrounding tissue was used for validation of the signatures. We were able to develop and validate a signature of methylation in HCC. This signature distinguished HCC from surrounding tissue and from other tumor types, and was independent of risk factors. However, aberrant methylation of an independent subset of promoters was associated with tumor progression and etiological risk factors (HBV or HCV infection and alcohol consumption). Interestingly, distinct methylation of an independent panel of gene promoters was strongly correlated with survival after cancer therapy. Conclusion Our study shows that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression stage, with potential clinical applications in diagnosis and prognosis.

Molecular profiling of docetaxel cytotoxicity in breast cancer cells: uncoupling of aberrant mitosis and apoptosis

Among microtubule-targeting agents, docetaxel has received recent interest owing to its good therapeutic index. Clinical trials have underlined its potential for the treatment of advanced breast cancer, although little is known about its molecular mode of action in this context. We characterized the molecular changes induced by docetaxel in two well-known human breast carcinoma cell lines. Two mechanisms of action according to drug concentration were suggested by a biphasic sensitivity curve, and were further validated by cell morphology, cell cycle and cell death changes. Two to four nanomolar docetaxel induced aberrant mitosis followed by late necrosis, and 100 nM docetaxel induced mitotic arrest followed by apoptosis. Passing through mitosis phase was a requirement for hypodiploidy to occur, as shown by functional studies in synchronized cells and by combining docetaxel with the proteasome inhibitor MG132. Transcriptional profiling showed differences according to cell line and docetaxel concentration, with cell cycle, cell death and structural genes commonly regulated in both cell lines. Although p53 targets were mainly induced with low concentration of drug in MCF7 cells, its relevance in the dual mechanism of docetaxel cytotoxicity was ruled out by using an isogenic shp53 cell line. Many of the genes shown in this study may contribute to the dual mechanism by which docetaxel inhibits the growth of breast cancer cells at different concentrations. These findings provide a basis for rationally enhancing docetaxel therapy, considering lower concentrations, and better drug combinations.

Modulation of DNA methylation states and infant immune system by dietary supplementation with ω-3 PUFA during pregnancy in an intervention study

BACKGROUND Early-life exposures to tobacco smoke and some dietary factors have been identified to induce epigenetic changes in genes involved in allergy and asthma development. Omega-3 (n-3) polyunsaturated fatty acid (PUFA) intake during pregnancy could modulate key cytokines and T helper (Th) cell maturation; however, little is known about the mechanism by which ω-3 PUFA could have a beneficial effect in preventing inflammatory disorders. OBJECTIVE We sought to test whether prenatal dietary supplementation with ω-3 PUFA during pregnancy may modulate epigenetic states in the infant immune system. DESIGN This study was based on a randomized intervention trial conducted in Mexican pregnant women supplemented daily with 400 mg docosahexaenoic acid (DHA) or a placebo from 18 to 22 wk of gestation to parturition. We applied quantitative profiling of DNA methylation states in Th1, Th2, Th17, and regulatory T-relevant genes as well as LINE1 repetitive elements of cord blood mononuclear cells (n = 261). RESULTS No significant difference in promoter methylation levels was shown between ω-3 PUFA-supplemented and control groups for the genes analyzed; however, ω-3 PUFA supplementation was associated with changes in methylation levels in LINE1 repetitive elements (P = 0.03) in infants of mothers who smoked during pregnancy. Furthermore, an association between the promoter methylation levels of IFNγ and IL13 was modulated by ω-3 PUFA supplementation (P = 0.06). CONCLUSIONS Our results indicate that maternal supplementation with ω-3 PUFA during pregnancy may modulate global methylation levels and the Th1/Th2 balance in infants. Therefore, the epigenetic mechanisms could provide attractive targets for prenatal modulation and prevention of inflammatory disorders and potentially other related diseases in childhood and adulthood.

MicroRNA miR-30 family regulates non-attachment growth of breast cancer cells

BackgroundA subset of breast cancer cells displays increased ability to self-renew and reproduce breast cancer heterogeneity. The characterization of these so-called putative breast tumor-initiating cells (BT-ICs) may open the road for novel therapeutic strategies. As microRNAs (miRNAs) control developmental programs in stem cells, BT-ICs may also rely on specific miRNA profiles for their sustained activity. To explore the notion that miRNAs may have a role in sustaining BT-ICs, we performed a comprehensive profiling of miRNA expression in a model of putative BT-ICs enriched by non-attachment growth conditions.ResultsWe found breast cancer cells grown under non-attachment conditions display a unique pattern of miRNA expression, highlighted by a marked low expression of miR-30 family members relative to parental cells. We further show that miR-30a regulates non-attachment growth. A target screening revealed that miR-30 family redundantly modulates the expression of apoptosis and proliferation-related genes. At least one of these targets, the anti-apoptotic protein AVEN, was able to partially revert the effect of miR-30a overexpression. Finally, overexpression of miR-30a in vivo was associated with reduced breast tumor progression.ConclusionsmiR30-family regulates the growth of breast cancer cells in non-attachment conditions. This is the first analysis of target prediction in a whole family of microRNAs potentially involved in survival of putative BT-ICs.

Independent genomewide screens identify the tumor suppressor VTRNA2-1 as a human epiallele responsive to periconceptional environment.

BackgroundInterindividual epigenetic variation that occurs systemically must be established prior to gastrulation in the very early embryo and, because it is systemic, can be assessed in easily biopsiable tissues. We employ two independent genome-wide approaches to search for such variants.ResultsFirst, we screen for metastable epialleles by performing genomewide bisulfite sequencing in peripheral blood lymphocyte (PBL) and hair follicle DNA from two Caucasian adults. Second, we conduct a genomewide screen for genomic regions at which PBL DNA methylation is affected by season of conception in rural Gambia. Remarkably, both approaches identify the genomically imprinted VTRNA2-1 as a top environmentally responsive epiallele. We demonstrate systemic and stochastic interindividual variation in DNA methylation at the VTRNA2-1 differentially methylated region in healthy Caucasian and Asian adults and show, in rural Gambians, that periconceptional environment affects offspring VTRNA2-1 epigenotype, which is stable over at least 10 years. This unbiased screen also identifies over 100 additional candidate metastable epialleles, and shows that these are associated with cis genomic features including transposable elements.ConclusionsThe non-coding VTRNA2-1 transcript (also called nc886) is a putative tumor suppressor and modulator of innate immunity. Thus, these data indicating environmentally induced loss of imprinting at VTRNA2-1 constitute a plausible causal pathway linking early embryonic environment, epigenetic alteration, and human disease. More broadly, the list of candidate metastable epialleles provides a resource for future studies of epigenetic variation and human disease.

Tobacco smoking-associated genome-wide DNA methylation changes in the EPIC study

AIM Epigenetic changes may occur in response to environmental stressors, and an altered epigenome pattern may represent a stable signature of environmental exposure. MATERIALS & METHODS Here, we examined the potential of DNA methylation changes in 910 prediagnostic peripheral blood samples as a marker of exposure to tobacco smoke in a large multinational cohort. RESULTS We identified 748 CpG sites that were differentially methylated between smokers and nonsmokers, among which we identified novel regionally clustered CpGs associated with active smoking. Importantly, we found a marked reversibility of methylation changes after smoking cessation, although specific genes remained differentially methylated up to 22 years after cessation. CONCLUSION Our study has comprehensively cataloged the smoking-associated DNA methylation alterations and showed that these alterations are reversible after smoking cessation.

Gene expression profiling of breast cancer cells in response to gemcitabine: NF-κB pathway activation as a potential mechanism of resistance

Gemcitabine is a nucleoside analog with clinical relevance in the treatment of several solid tumors, including breast carcinoma. In spite of its cytotoxic effect, clinical efficacy is impaired by the development of resistance. We performed gene expression analysis to shed light into the molecular mechanism of action of this drug in two breast cancer cell lines. Activation of genes related with cell cycle, cell growth and apoptosis (BNIP3L, CCNG2, DDIT4, TGFB2, TP53BP1, TP53INP1, and VEGF) was the main finding in the p53-wild type cell line MCF7, while the p53-non-functional cell line MDA-MB-231 was characterized by the regulation of NF-κB target genes (BIRC3, CXCL1/GRO1, IRAK2, TNF, TNFAIP and TRAF1). Genes consistently induced (ATF3, CCNG2, CDKN1A, EGR1, INSIG1, and MAF) or repressed (CCND1 and VGF) in both cell lines, were also found after gemcitabine treatment. In addition, MDA-MB-231 cells showed a higher basal and induced NF-κB transcriptional activity after treatment with gemcitabine. In comparison with gemcitabine, gene expression after 5-fluorouracil treatment showed essentially different profiles in both cell lines. This, in spite of using equitoxic concentrations producing similar effects on cell cycle. NF-κB transcriptional activity in MDA-MB-231 cells was dependent on IκB-alpha phosphorylation, as shown by functional experiments using the specific inhibitor BAY11-7082. Moreover, immunohistochemical analysis of clinical samples of breast carcinoma further validated the induction of NF-κB expression and IκB down-regulation upon neoadjuvant gemcitabine treatment. Thus, gene expression patterns, in vitro functional studies and analysis of tissue samples are in agreement with a role for NF-κB pathway in gemcitabine response. Together with the reported role for NF-κB in the induction of resistance to chemotherapy, our data gives support to clinical strategies combining gemcitabine with NF-κB inhibitors in breast cancer.

DNA methylation in peripheral blood measured by LUMA is associated with breast cancer in a population-based study

Our purpose was to identify epigenetic markers of breast cancer risk, which can be reliably measured in peripheral blood and are amenable for large population screening. We used 2 independent assays, luminometric methylation assay (LUMA) and long interspersed elements‐1 (LINE‐1) to measure “global methylation content” in peripheral blood DNA from a well‐characterized population‐based case‐control study. We examined associations between methylation levels and breast cancer risk among 1055 cases and 1101 controls and potential influences of 1‐carbon metabolism on global methylation. Compared with women in the lowest quintile of LUMA methylation, those in the highest quintile had a 2.41‐fold increased risk of breast cancer (95% confidence interval: 1.83‐3.16; P, trend>0.0001). The association did not vary by other key tumor characteristics and lifestyle risk factors. Consistent with LUMA findings, genome‐wide methylation profiling of a subset of samples revealed greater promoter hypermethylation in breast cancer case participants (P=0.04); higher LUMA was associated with higher promoter methylation in the controls (P=0.05). LUMA levels were also associated with functional sodium nitroprusside in key 1‐carbon metabolizing genes, MTHFR C677T (P=0.001) and MTRR A66G (P=0.018). LINE‐1 methylation was associated with neither breast cancer risk nor 1‐carbon metabolism. Our results show that global promoter hypermethylation measured in peripheral blood was associated with breast cancer risk.—Xu, X., Gammon, M. D., Hernandez‐Vargas, H., Herceg, Z., Wetmur, J. G., Teitelbaum, S. L., Bradshaw, P. T., Neugut, A. I., Santella, R. M., Chen, J. DNA methylation in peripheral blood measured by LUMA is associated with breast cancer in a population‐based study. FASEB J. 26, 2657‐2666 (2012). www.fasebj.org

Transcriptional profiling of MCF7 breast cancer cells in response to 5‐Fluorouracil: Relationship with cell cycle changes and apoptosis, and identification of novel targets of p53

The availability of oral precursors of 5‐Fluorouracil (5‐FU) and its favorable results in treating advanced breast cancer have renewed the interest in the molecular mechanisms underlying its cytotoxicity. We have compared the changes in cell cycle and cell death parameters induced by 2 different concentrations of 5‐FU (IC50 and IC80) in the breast adenocarcinoma cell line MCF7. G1/S cell cycle arrest was associated with both concentrations, whereas cell death was mainly induced after IC80 5‐FU. These changes were correlated with gene expression assessed by cDNA microarray analysis. Main findings included an overexpression of p53 target genes involved in cell cycle and apoptosis (CDKN1A/p21, TP53INP, TNFRSF6/FAS and BBC3/PUMA), and significant repression of Myc. High dose 5‐FU also induced a higher regulation of the mitochondrial death genes APAF1, BAK1 and BCL2, and induction of genes of the ID family. Furthermore, we establish a direct causal relationship between p21, ID1 and ID2 overexpression, increased acetylation of histones H3 and H4 and binding of p53 to their promoters as a result of 5‐FU treatment. The relevance of these findings was further studied after interfering p53 expression in MCF7 cells (shp53 cells), showing a lower induction of both, ID1 and ID2 transcripts, after 5‐FU when compared with MCF7 shGFP control cells. This molecular characterization of dose‐ and time‐dependent modifications of gene expression after 5‐FU treatment should provide a resource for future basic studies addressing the molecular mechanisms of chemotherapy in breast cancer.

Methylome analysis reveals Jak-STAT pathway deregulation in putative breast cancer stem cells.

Growing evidence supports the existence of a subpopulation of cancer cells with stem cell characteristics within breast tumors. In spite of its potential clinical implications, an understanding of the mechanisms responsible for retaining the stem cell characteristics in these cells is still lacking. Here, we used the mammosphere model combined with DNA methylation bead arrays and quantitative gene expression to characterize the epigenetic mechanisms involved in the regulation of developmental pathways in putative breast cancer stem cells. Our results revealed that MCF7-derived mammospheres exhibit distinct CpG promoter methylation profiles in a specific set of genes, including those involved in Jak-STAT signaling pathway. Hypomethylation of several gene components of the Jak-STAT pathway was correlated with an increased expression in mammospheres relative to parental cells. Remarkably, cell sorting of the cells with a putative cancer stem cell phenotype (CD44+/CD24 low) suggests a constitutive activation of Jak-STAT pathway in these cells. These results show that Jak-STAT activation may represent a characteristic of putative breast cancer stem cells. In addition, they favor the concept that the expression of cancer stem-like pathways and the establishment and maintenance of defining properties of cancer stem cells are orchestrated by epigenetic mechanisms.