Transcriptional epigenetic regulation of Fkbp1/Pax9 genes is associated with impaired sensitivity to platinum treatment in ovarian cancer

Background: In an effort to contribute to overcoming the platinum resistance exhibited by most solid tumors, we performed an array of epigenetic approaches, integrating next-generation methodologies and public clinical data to identify new potential epi-biomarkers in ovarian cancer, which is considered the most devastating of gynecological malignancies. Methods: We cross-analyzed data from methylome assessments and restoration of gene expression through microarray expression in a panel of four paired cisplatin-sensitive/cisplatin-resistant ovarian cancer cell lines, along with publicly available clinical data from selected individuals representing the state of chemoresistance. We validated the methylation state and expression levels of candidate genes in each cellular phenotype through Sanger sequencing and reverse transcription polymerase chain reaction, respectively. We tested the biological role of selected targets using an ectopic expression plasmid assay in the sensitive/resistant tumor cell lines, assessing the cell viability in the transfected groups. Epigenetic features were also assessed in 189 primary samples obtained from ovarian tumors and controls. Results: We identified PAX9 and FKBP1B as potential candidate genes, which exhibited epigenetic patterns of expression regulation in the experimental approach. Re-establishment of FKBP1B expression in the resistant OVCAR3 phenotype in which this gene is hypermethylated and inhibited allowed it to achieve a degree of platinum sensitivity similar to the sensitive phenotype. The evaluation of these genes at a translational level revealed that PAX9 hypermethylation leads to a poorer prognosis in terms of overall survival. We also set a precedent for establishing a common epigenetic signature in which the validation of a single candidate, MEST, proved the accuracy of our computational pipelines. Conclusions: Epigenetic regulation of PAX9 and FKBP1B genes shows that methylation in non-promoter areas has the potential to control gene expression and thus biological consequences, such as the loss of platinum sensitivity. At the translational level, PAX9 behaves as a predictor of chemotherapy response to platinum in patients with ovarian cancer. This study revealed the importance of the transcript-specific study of each gene under potential epigenetic regulation, which would favor the identification of new markers capable of predicting each patient’s progression and therapeutic response.The study was financially supported by FIS (ISCIII) and ERDF/FSE funds (PI15/00186, PI18/0050, and ERDF/FSE, A way to make Europe). The authors gratefully acknowledge the Colombian Ministry for Science, Technology and Innovation (MINCIENCIAS), Code 568-2012, for providing J.S. with partial funding for this study

A novel role for the tumor suppressor gene itf2 in tumorigenesis and chemotherapy response

Despite often leading to platinum resistance, platinum-based chemotherapy continues to be the standard treatment for many epithelial tumors. In this study we analyzed and validated the cytogenetic alterations that arise after treatment in four lung and ovarian paired cisplatin-sensitive/resistant cell lines by 1-million microarray-based comparative genomic hybridization (array-CGH) and qRT-PCR methodologies. RNA-sequencing, functional transfection assays, and gene-pathway activity analysis were used to identify genes with a potential role in the development of this malignancy. The results were further explored in 55 lung and ovarian primary tumors and control samples, and in two extensive in silico databases. Long-term cell exposure to platinum induces the frequent deletion of ITF2 gene. Its expression re-sensitized tumor cells to platinum and recovered the levels of Wnt/β-catenin transcriptional activity. ITF2 expression was also frequently downregulated in epithelial tumors, predicting a worse overall survival. We also identified an inverse correlation between ITF2 and HOXD9 expression, revealing that Non-small cell lung cancer (NSCLC) patients with lower expression of HOXD9 had a better overall survival rate. We defined the implication of ITF2 as a molecular mechanism behind the development of cisplatin resistance probably through the activation of the Wnt-signaling pathway. This data highlights the possible role of ITF2 and HOXD9 as novel therapeutic targets for platinum resistant tumors.This research was funded by the Fondo de Investigación Sanitaria-Instituto de Salud Carlos III, PI15/00186 and PI18/00050, CP19/00063, and CM19/00100 for HR and by MINECO, RTC-2016-5314-1 to I.I.C; by the MINECO, SAF2016-75531-R, by the CAM B2017/BMD-3724 and by the AECC GCB14142311CRES to P.S; and the European Regional Development Fund/European Social Fund FIS (FEDER/FSE, Una Manera de Hacer Europa)

Original Article Biomarkers of erlotinib response in non-small cell lung cancer tumors that do not harbor the more common epidermal growth factor receptor mutations

Abstract: Non-small cell lung cancer (NSCLC) represents approximately 85% of all lung cancers, which are the leading cause of cancer-related deaths in the world. Tyrosine kinase inhibitors such as erlotinib represent one therapeutic options presently recommended for tumors produced by activating mutations in the gene coding of epidermal growth factor receptor (EGFR). The aim of this study is the identification of possible biomarkers for tumor sensitivity to erlotinib in the absence of the main EGFR mutations. The erlotinib sensitivity of cells isolated from 41 untreated NSCLC patients was determined and compared with the presence of the more frequent EGFR mutations. Several patients had tumor cells highly sensitive to erlitinib in the absence of the EGFR mutations analyzed. The gene expression profile of 3 erlotinib-sensitive tumors was compared with that of 4 resistant tumors by DNA microarray hybridization. Sixteen genes were expressed at significantly higher levels in the resistant tumors than in the sensitive tumors. The possible correlation between erlotinib sensitivity and the expression of these genes was further analyzed using the data for the NSCLC, breast cancer and colon cancer cell lines of the NCI60 collection. The expression of these genes was correlated with the overall survival of 5 patients treated with erlotinib, according to The Cancer Genome Atlas (TCGA) database. Overlapping groups of 7, 5 and 3 genes, including UGT1A6, TRIB3, MET, MMP7, COL17A1, LCN2 and PTPRZ1, whose expression correlated with erlotinib activity was identified. In particular, low MET expression levels showed the strongest correlation

Estudio de Línea Base de las Concentraciones de Metales Pesados en Chocolate en Polvo Comercializado en Guayaquil, Ecuador

Heavy metal contamination has been demonstrated in cocoa in Ecuador. The impact of this contamination on powdered chocolate for national consumption has not, however, been studied. This study's objective was to generate a baseline of arsenic, cadmium, and lead concentration in powdered chocolate marketed in Guayaquil. To execute this study, chocolate samples were acquired in the north, center, and south of the city in stores and supermarkets. The three most popular brands were chosen and analyzed by atomic absorption spectrophotometry with a graphite furnace. Lead was not detectable in the chocolate; arsenic was found at low levels in Brand-1 (0.030 ± 0.021 mg / kg) and in Brand-3 (0.060 ± 0.034 mg / kg). On the contrary, cadmium was found in all chocolate brands, with concentrations of 0.236 ± 0.082 mg/kg in Brand-1 and 0.169 ± 0.066 mg/kg in Brand-2.  Brand-3 exceeded the maximum permissible limit of 0.600 mg/kg according to European regulations with 1.440 ± 0.212 mg/kg, representing a risk for consumers.     En Ecuador se ha demostrado contaminación por metales pesados en el cacao; sin embargo, no se ha estudiado el impacto de esta contaminación sobre el chocolate en polvo de consumo nacional. El objetivo de este estudio fue generar una línea base de la concentración de arsénico, cadmio y plomo en chocolate en polvo comercializado en la ciudad de Guayaquil. Para ello, se adquirieron muestras de chocolate en las zonas norte, centro y sur de la ciudad en tiendas y supermercados. Se eligieron tres marcas más populares y se analizaron por espectrofotometría de absorción atómica con horno de grafito. El plomo no fue detectable en el chocolate; el As se encontró en bajos niveles en la Marca-1 (0,030 ± 0,021 mg/kg) y la Marca-3 (0,060 ± 0,034 mg/kg), y, por el contrario, se halló cadmio en todas las marcas de chocolate, con concentraciones de 0,236 ± 0,082 mg/kg en la Marca-1, 0,169 ± 0,066 mg/kg en la Marca-2 y la Marca-3 superó el límite máximo permisible de 0.600 mg/kg según normativa europea con 1,440 ± 0,212 mg/kg, representando un riesgo para los consumidores.

Metformin: A new option in cancer treatment

Metformin is a biguanine, the most widely used antidiabetic drug for the treatment of type 2 diabetes. Some studies suggest that metformin decreases the incidence of cancer and cancer-related mortality in diabetic patients. Metformin activates the AMP-activated protein kinase (AMPK) pathway, a major sensor of the energy status of the cell and an inhibitor of mammalian target of rapamycin (mTOR) catalytic activity, inducing a decrease in blood glucose by decreasing hepatic gluconeogenesis and stimulating glucose uptake in the muscle. Some preclinical data supports the inhibition of tumour cancer cell growth associated with mTOR inhibition and a decrease in phosphorylation of S6K, rpS6 and 4E-BP1. Here we have summarised some of the preclinical data and data of many clinical trials that are exploring the true value of metformin for cancer patients, mainly breast and prostate cancer. © 2011 Feseo.Peer Reviewe

Expression of the SERPING1 gene is not regulated by promoter hypermethylation in peripheral blood mononuclear cells from patients with hereditary angioedema due to C1-inhibitor deficiency

SERPING1 mutations causing Hereditary Angioedema type I (HAE-I) due to C1-Inhibitor (C1-INH) deficiency display a dominant-negative effect usually resulting in protein levels far below the expected 50%. To further investigate mechanisms for its reduced expression, we analyzed the promoter DNA methylation status of SERPING1 and its influence on C1-INH expression. Global epigenetic reactivation correlated with C1-INH mRNA synthesis and protein secretion in Huh7 hepatoma cells. However, PBMCs extracted from controls, HAE-I and HAE-II patients presented identical methylation status of the SERPING1 promoter when analyzed by bisulphite sequencing; the proximal CpG island (exon 2) is constitutively unmethylated, while the most distant one (5.7Kb upstream the transcriptional start site) is fully methylated. These results correlate with the methylation profile observed in Huh7 cells and indicate that there is not a direct epigenetic regulation of C1-INH expression in PBMCs specific for each HAE type. Other indirect modes of epigenetic regulation cannot be excludedThe work was funded by grants PS09/00122 by Instituto de Salud Carlos III (ISCIII) and Ministerio de Economía y Competitividad SAF2012-38636. OP and IIC were supported by FIS PS09/00472, PI12/00386 and Miguel Servet (CP 08/000689; PI-717). ALL was supported by CIBERER (Instituto de Salud Carlos III, ISCIII) and co-financed with FEDER fund

Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivo after peripheral inflammation: Potential role of microglial ERs

It has been previously reported that the neuroprotective hormone oestradiol reduces microglia inflammatory activity. The objective of this study was to test whether two selective oestrogen receptor modulators, tamoxifen and raloxifene, modulate in vivo the activation of microglia induced by the peripheral administration of lipopolysaccharide (LPS). Activation of microglia was assessed in the white matter of the cerebellum using immunoreactivity for major histocompatability complex-II. Oestradiol, tamoxifen and raloxifene decreased microglia activation induced by LPS in male and ovariectomized female rats, although the doses of oestradiol that were effective in decreasing microglia reactivity were not the same in both sexes. Tamoxifen reduced microglia activation in all experimental groups at all doses tested (0.5-2 mg/kg b.w.) while raloxifene lost its anti-inflammatory activity at the higher dose tested (2 mg/kg b.w). In addition, raloxifene had per se a moderate pro-inflammatory activity in the brain of control female rats and its anti-inflammatory activity was partially impaired in female animals after 1 month of deprivation of ovarian hormones. Spots of oestrogen receptor (ER)-α immunoreactivity were detected in the soma and cell processes of microglia. Treatment with LPS, oestradiol or tamoxifen induced an increase of ER-α immunoreactive spots in the perikaryon of microglia, while oestradiol antagonized the effect of LPS. The results indicate that some oestrogenic compounds decrease brain inflammation by a mechanism that may involve ERs expressed by microglia. The findings support the potential therapeutic role of oestrogenic compounds as protective anti-inflammatory agents for the central nervous system. © 2008 Society for Endocrinology.Peer Reviewe

Pubertal maturation modifies the regulation of insulin-like growth factor-I receptor signaling by estradiol in the rat prefrontal cortex

The transition from adolescence to adulthood is accompanied by substantial plastic modifications in the cerebral cortex, including changes in the growth and retraction of neuronal processes and in the rate of synaptic formation and neuronal loss. Some of these plastic changes are prevented in female rats by prepubertal ovariectomy. The ovarian hormone estradiol modulates neuronal differentiation and survival and these effects are in part mediated by the interaction with insulin-like growth factor-I (IGF-I). In this study, we have explored whether the activation by estradiol of some components of IGF-I receptor signaling is altered in the prefrontal cortex during puberty. Estradiol administration to rats ovariectomized after puberty resulted, 24 h after the hormonal administration, in a sustained phosphorylation of Akt and glycogen synthase kinase 3β in the prefrontal cortex. However, this hormonal effect was not observed in animals ovariectomized before puberty. These findings suggest that during pubertal maturation there is a programming by ovarian hormones of the future regulatory actions of estradiol on IGF-I receptor signaling in the prefrontal cortex. The modification in the regulation of IGF-I receptor signaling by estradiol during pubertal maturation may have implications for the developmental changes occurring in the prefrontal cortex in the transition from adolescence to adulthood. © 2008 Wiley Periodicals, Inc.Peer Reviewe

Selective estrogen receptor modulators decrease reactive astrogliosis in the injured brain: Effects of aging and prolonged depletion of ovarian hormones

After brain injury, astrocytes acquire a reactive phenotype characterized by a series of morphological and molecular modifications, including the expression of the cytoskeletal protein vimentin. Previous studies have shown that estradiol down-regulates reactive astrogliosis. In this study we assessed whether raloxifene and tamoxifen, two selective estrogen receptor modulators, have effects similar to estradiol in astrocytes. We also assessed whether aging and the timing of estrogenic therapy after ovariectomy influence the action of the estrogenic compounds. Four groups of animals were studied: 1) young rats, ovariectomized at 2 months of age; 2) middle-aged rats, ovariectomized at 8 months of age; 3) aged rats, ovariectomized at 18 months of age; and 4) aged rats, ovariectomized at 2 months and sham operated at 18 months of age. Fifteen days after ovariectomy or sham surgery, animals received a stab wound brain injury and the treatment with the estrogenic compounds. The number of vimentin-immunoreactive astrocytes after injury was significantly higher in the hippocampus of aged rats after a long-term ovariectomy compared with aged animals after a short-term ovariectomy and middle-aged rats. In addition, reactive astrocytes were more numerous in the two groups of aged animals than in young animals. Despite these differences, the estrogenic compounds reduced reactive astrogliosis in all animal groups. These findings indicate that estradiol, raloxifene, and tamoxifen are potential candidates for the control of astrogliosis in young and older individuals and after a prolonged depletion of ovarian hormones. Copyright © 2009 by The Endocrine Society.Peer Reviewe

Methylation status of IGFBP-3 as a useful clinical tool for deciding on a concomitant radiotherapy

Olga Pernía et al.© 2014 Taylor & Francis Group, LLC. The methylation status of the IGFBP-3 gene is strongly associated with cisplatin sensitivity in patients with non-small cell lung cancer (NSCLC). In this study, we found in vitro evidence that linked the presence of an unmethylated promoter with poor response to radiation. Our data also indicate that radiation might sensitize chemotherapy-resistant cells by reactivating IGFBP-3-expression through promoter demethylation, inactivating the PI3K/AKT pathway. We also explored the IGFBP-3 methylation effect on overall survival (OS) in a population of 40 NSCLC patients who received adjuvant therapy after R0 surgery. Our results indicate that patients harboring an unmethylated promoter could benefit more from a chemotherapy schedule alone than from a multimodality therapy involving radiotherapy and platinum-based treatments, increasing their OS by 2.5 y (p =.03). Our findings discard this epi-marker as a prognostic factor in a patient population without adjuvant therapy, indicating that radiotherapy does not improve survival for patients harboring an unmethylated IGFBP-3 promoter.Supported by FIS (ISCIII), PI12/00386, PI12/01463, PI11/00949; PI11/00537; PTA2012/7141-I, all supported by FEDER funds. IIC was supported by the “Miguel Servet” program (CP 08/000689)Peer Reviewe