Transcriptome profile analysis reveals putative molecular mechanisms of 5-aminolevulinic acid toxicity.

5-aminolevulinic acid (5-ALA) is the first precursor of the heme biosynthesis pathway, accumulated in acute intermittent porphyria (AIP), an inherited metabolic disease characterized by porphobilinogen deaminase deficiency. An increased incidence of hepatocellular carcinoma (HCC) has been reported as a long-term manifestation in symptomatic AIP patients. 5-ALA is an α-aminoketone prone to oxidation, yielding reactive oxygen species and 4,5-dioxovaleric acid. A high concentration of 5-ALA presents deleterious pro-oxidant potential. It can induce apoptosis, DNA damage, mitochondrial dysfunction, and altered expression of carcinogenesis-related proteins. Several hypotheses of the increased risk of HCC rely on the harmful effect of elevated 5-ALA in the liver of AIP patients, which could promote a pro-carcinogenic environment. We investigated the global transcriptional changes and perturbed molecular pathways in HepG2 cells following exposure to 5-ALA 25 mM for 2 h and 24 h using DNA microarray. Distinct transcriptome profiles were observed. 5-ALA '25 mM-2h' upregulated 10 genes associated with oxidative stress response and carcinogenesis. Enrichment analysis of differentially expressed genes by KEGG, Reactome, MetaCore™, and Gene Ontology, showed that 5-ALA '25 mM-24h' enriched pathways involved in drug detoxification, oxidative stress, DNA damage, cell death/survival, cell cycle, and mitochondria dysfunction corroborating the pro-oxidant properties of 5-ALA. Furthermore, our results disclosed other possible processes such as senescence, immune responses, endoplasmic reticulum stress, and also some putative effectors, such as sequestosome, osteopontin, and lon peptidase 1. This study provided additional knowledge about molecular mechanisms of 5-ALA toxicity which is essential to a deeper understanding of AIP and HCC pathophysiology. Furthermore, our findings can contribute to improving the efficacy of current therapies and the development of novel biomarkers and targets for diagnosis, prognosis, and therapeutic strategies for AHP/AIP and associated HCC.

Bradykinin promotes immune responses in differentiated embryonic neurospheres carrying APPswe and PS1dE9 mutations.

BACKGROUND: Neural progenitor cells (NPCs) can be cultivated from developing brains, reproducing many of the processes that occur during neural development. They can be isolated from a variety of animal models, such as transgenic mice carrying mutations in amyloid precursor protein (APP) and presenilin 1 and 2 (PSEN 1 and 2), characteristic of familial Alzheimer's disease (fAD). Modulating the development of these cells with inflammation-related peptides, such as bradykinin (BK) and its antagonist HOE-140, enables the understanding of the impact of such molecules in a relevant AD model. RESULTS: We performed a global gene expression analysis on transgenic neurospheres treated with BK and HOE-140. To validate the microarray data, quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed on 8 important genes related to the immune response in AD such as CCL12, CCL5, CCL3, C3, CX3CR1, TLR2 and TNF alpha and Iba-1. Furthermore, comparative analysis of the transcriptional profiles was performed between treatments, including gene ontology and reactome enrichment, construction and analysis of protein-protein interaction networks and, finally, comparison of our data with human dataset from AD patients. The treatments affected the expression levels of genes mainly related to microglia-mediated neuroinflammatory responses, with BK promoting an increase in the expression of genes that enrich processes, biological pathways, and cellular components related to immune dysfunction, neurodegeneration and cell cycle. B2 receptor inhibition by HOE-140 resulted in the reduction of AD-related anomalies caused in this system. CONCLUSIONS: BK is an important immunomodulatory agent and enhances the immunological changes identified in transgenic neurospheres carrying the genetic load of AD. Bradykinin treatments modulate the expression rates of genes related to microglia-mediated neuroinflammation. Inhibiting bradykinin activity in Alzheimer's disease may slow disease progression.

Differentiated Embryonic Neurospheres from Familial Alzheimer's Disease Model Show Innate Immune and Glial Cell Responses.

Proteins involved in the Alzheimer's disease (AD), such as amyloid precursor protein (APP) and presenilin-1 (PS1), play critical roles in early development of the central nervous system (CNS), as well as in innate immune and glial cell responses. Familial AD is associated with the presence of APPswe and PS1dE9 mutations. However, it is still unknown whether these mutations cause deficits in CNS development of carriers. We studied genome-wide gene expression profiles of differentiated neural progenitor cells (NPCs) from wild-type and APPswe/PS1dE9 mouse embryo telencephalon. The occurrence of strong innate immune and glial cell responses in APPswe/PS1dE9 neurospheres mainly involves microglial activation, inflammatory mediators and chemokines. APPswe/PS1dE9 neurospheres augmented up to 100-fold CCL12, CCL5, CCL3, C3, CX3CR1, TLR2 and TNF-alpha expression levels, when compared to WT neurospheres. Expression levels of the glia cell marker GFAP and microglia marker Iba-1 were up to 20-fold upregulated in APPswe/PS1dE9 neurospheres. The secretome of differentiated APPswe/PS1dE9 NPCs revealed enhanced chemoattraction of peripheral blood mononuclear cells. When evaluating the inferred protein interaction networks constructed from the array data, an improvement in astrocyte differentiation in APPswe/PS1dE9 neurospheres was evident in view of increased GFAP expression. Transgenic NPCs differentiated into neural phenotypes presented expression patterns of cytokine, glial cells, and inflammatory mediators characteristic of APPswe/PS1dE9 adult animals. Consequently, the neurogenic niche obtained from differentiation of embryonic APPswe/PS1dE9 neurospheres spontaneously presents several alterations observed in adult AD brains. Finally, our data strengthen pathophysiological hypotheses that propose an early neurodevelopmental origin for familial AD.

DNA methylation of the promoter region at the CREB1 binding site is a mechanism for the epigenetic regulation of brain-specific PKMζ.

Protein kinase M zeta, PKMζ, is a brain enriched kinase with a well characterized role in Long-Term Potentiation (LTP), the activity-dependent strengthening of synapses involved in long-term memory formation. However, little is known about the molecular mechanisms that maintain the tissue specificity of this kinase. Here, we characterized the epigenetic factors, mainly DNA methylation, regulating PKMζ expression in the human brain. The PRKCZ gene has an upstream promoter regulating Protein kinase C ζ (PKCζ), and an internal promoter driving PKMζ expression. A demethylated region, including a canonical CREB binding site, situated at the internal promoter was only observed in human CNS tissues. The induction of site-specific hypermethylation of this region resulted in decreased CREB1 binding and downregulation of PKMζ expression. Noteworthy, CREB binding sites were absent in the upstream promoter of PRKCZ locus, suggesting a specific mechanism for regulating PKMζ expression. These observations were validated using a system of human neuronal differentiation from induced pluripotent stem cells (iPSCs). CREB1 binding at the internal promoter was detected only in differentiated neurons, where PKMζ is expressed. The same epigenetic mechanism in the context of CREB binding site was identified in other genes involved in neuronal differentiation and LTP. Additionally, aberrant DNA hypermethylation at the internal promoter was observed in cases of Alzheimer's disease, correlating with decreased expression of PKMζ in patient brains. Altogether, we present a conserved epigenetic mechanism regulating PKMζ expression and other genes enhanced in the CNS with possible implications in neuronal differentiation and Alzheimer's disease.

Prognostic value of integrin αV expression and localization pattern in invasive breast carcinomas.

Invasion of surrounding stroma is an early event in breast cancer metastatic progression, and involves loss of cell polarity, loss of myoepithelial layer, epithelial-mesenchymal transition (EMT) and remodeling of the extracellular matrix (ECM). Integrins are transmembrane receptors responsible for cell-ECM binding, which triggers signals that regulate many aspects of cell behavior and fate. Changes in the expression, localization and pairing of integrins contribute for abnormal responses found in transformed epithelia. We analyzed 345 human breast cancer samples in tissue microarrays (TMA) from cases diagnosed with invasive breast carcinoma to assess the expression and localization pattern of integrin αV and correlation with clinical parameters. Patients with lower levels of integrin αV staining showed reduced cancer specific survival. A subset of cases presented a peripheral staining of integrin αV surrounding tumor cell clusters, possibly matching the remaining myoepithelial layer. Indeed, the majority of ductal carcinoma in situ (DCIS) components found in the TMA presented integrin αV at their periphery, whereas this pattern was mostly lost in invasive components, even in the same sample. The lack of peripheral integrin αV correlated with decreased cancer specific survival. In addition, we observed that the presence of integrin αV in the stroma was an indicative of poor survival and metastatic disease. Consistently, by interrogating publicly available datasets we found that, although patients with higher mRNA levels of integrin αV had increased risk of developing metastasis, high co-expression of integrin αV and a myoepithelial cell marker (MYH11) mRNA levels correlated with better clinical outcomes. Finally, a 3D cell culture model of non-malignant and malignant cells reproduced the integrin αV pattern seen in patient samples. Taken together, our data indicate that both the expression levels of integrin αV and its tissue localization in primary tumors have prognostic value, and thus, could be used to help predict patients at higher risk of developing metastasis.

Genes regulated by DNA methylation are involved in distinct phenotypes during melanoma progression and are prognostic factors for patients.

In addition to mutations, epigenetic alterations are important contributors to malignant transformation and tumor progression. The aim of this work was to identify epigenetic events in which promoter or gene body DNA methylation induces gene expression changes that drive melanocyte malignant transformation and metastasis. We previously developed a linear mouse model of melanoma progression consisting of spontaneously immortalized melanocytes, premalignant melanocytes, a nonmetastatic tumorigenic, and a metastatic cell line. Here, through the integrative analysis of methylome and transcriptome data, we identified the relationship between promoter and/or gene body DNA methylation alterations and gene expression in early, intermediate, and late stages of melanoma progression. We identified adenylate cyclase type 3 (Adcy3) and inositol polyphosphate 4-phosphatase type II (Inpp4b), which affect tumor growth and metastatic potential, respectively. Importantly, the gene expression and DNA methylation profiles found in this murine model of melanoma progression were correlated with available clinical data from large population-based primary melanoma cohorts, revealing potential prognostic markers.

Transcriptional signatures underlying dynamic phenotypic switching and novel disease biomarkers in a linear cellular model of melanoma progression.

Despite advances in therapeutics, the progression of melanoma to metastasis still confers a poor outcome to patients. Nevertheless, there is a scarcity of biological models to understand cellular and molecular changes taking place along disease progression. Here, we characterized the transcriptome profiles of a multi-stage murine model of melanoma progression comprising a nontumorigenic melanocyte lineage (melan-a), premalignant melanocytes (4C), nonmetastatic (4C11-) and metastasis-prone (4C11+) melanoma cells. Clustering analyses have grouped the 4 cell lines according to their differentiated (melan-a and 4C11+) or undifferentiated/"mesenchymal-like" (4C and 4C11-) morphologies, suggesting dynamic gene expression patterns associated with the transition between these phenotypes. The cell plasticity observed in the murine melanoma progression model was corroborated by molecular markers described during stepwise human melanoma differentiation, as the differentiated cell lines in our model exhibit upregulation of transitory and melanocytic markers, whereas "mesenchymal-like" cells show increased expression of undifferentiated and neural crest-like markers. Sets of differentially expressed genes (DEGs) were detected at each transition step of tumor progression, and transcriptional signatures related to malignancy, metastasis and epithelial-to-mesenchymal transition were identified. Finally, DEGs were mapped to their human orthologs and evaluated in uni- and multivariate survival analyses using gene expression and clinical data of 703 drug-naïve primary melanoma patients, revealing several independent candidate prognostic markers. Altogether, these results provide novel insights into the molecular mechanisms underlying the phenotypic switch taking place during melanoma progression, reveal potential drug targets and prognostic biomarkers, and corroborate the translational relevance of this unique sequential model of melanoma progression.

Aurora A kinase and its activator TPX2 are potential therapeutic targets in KRAS-induced pancreatic cancer.

PURPOSE: Oncogenic KRAS mutations are found in over 90% of pancreatic ductal adenocarcinomas (PDACs). As yet, however, no effective therapies are available for KRAS-induced malignancies. Therefore, research aimed at the identification of KRAS targets with therapeutic potential is warranted. Our goal was to investigate Aurora A (AURKA) and targeting protein for Xklp2 (TPX2) as potential therapeutic targets in PDAC. METHODS: AURKA and TPX2 expression was assessed using RNAseq and qRT-PCR in PDAC patient samples and matched non-tumor pancreatic tissues. Publicly available PDAC datasets were used to investigate associations of AURKA and TPX2 expression levels with patient survival and the presence of KRAS mutations. Next, we used an Aurora kinase inhibitor, or KRAS, AURKA and TPX2 targeting using RNA interference in KRAS-mutant PDAC cells and, subsequently, analyzed their clonogenic and anchorage-independent growth and migration. RESULTS: We found that relative to matched non-tumor tissues, PDAC tumors displayed significantly higher expression levels of AURKA and TPX2. In addition, we found that AURKA and TPX2 were co-expressed in PDAC datasets, and that high expression levels of AURKA and TPX2 were associated with a shorter patient survival and with the presence of oncogenic KRAS mutations. In addition, we found that siRNA-mediated KRAS targeting in KRAS-mutant PDAC cells reduced AURKA and TPX2 expression. Furthermore, targeting AURKA or TPX2 in KRAS-mutant PDAC cells reduced their clonogenic and anchorage-independent growth, as well their migration. CONCLUSIONS: From our data we conclude that AURKA and TPX2 may act as KRAS biomarkers in PDAC that can predict a worse prognosis, and that AURKA or TPX2 targeting in PDAC cells may reduce their transformed phenotype. These results indicate that AURKA and TPX2 may serve as promising targets to be explored for KRAS-mutant PDAC therapy.

Gene co-expression and histone modification signatures are associated with melanoma progression, epithelial-to-mesenchymal transition, and metastasis.

BACKGROUND: We have previously developed a murine cellular system that models the transformation from melanocytes to metastatic melanoma cells. This model was established by cycles of anchorage impediment of melanocytes and consists of four cell lines: differentiated melanocytes (melan-a), pre-malignant melanocytes (4C), malignant (4C11-), and metastasis-prone (4C11+) melanoma cells. Here, we searched for transcriptional and epigenetic signatures associated with melanoma progression and metastasis by performing a gene co-expression analysis of transcriptome data and a mass-spectrometry-based profiling of histone modifications in this model. RESULTS: Eighteen modules of co-expressed genes were identified, and some of them were associated with melanoma progression, epithelial-to-mesenchymal transition (EMT), and metastasis. The genes in these modules participate in biological processes like focal adhesion, cell migration, extracellular matrix organization, endocytosis, cell cycle, DNA repair, protein ubiquitination, and autophagy. Modules and hub signatures related to EMT and metastasis (turquoise, green yellow, and yellow) were significantly enriched in genes associated to patient survival in two independent melanoma cohorts (TCGA and Leeds), suggesting they could be sources of novel prognostic biomarkers. Clusters of histone modifications were also linked to melanoma progression, EMT, and metastasis. Reduced levels of H4K5ac and H4K8ac marks were seen in the pre-malignant and tumorigenic cell lines, whereas the methylation patterns of H3K4, H3K56, and H4K20 were related to EMT. Moreover, the metastatic 4C11+ cell line showed higher H3K9me2 and H3K36me3 methylation, lower H3K18me1, H3K23me1, H3K79me2, and H3K36me2 marks and, in agreement, downregulation of the H3K36me2 methyltransferase Nsd1. CONCLUSIONS: We uncovered transcriptional and histone modification signatures that may be molecular events driving melanoma progression and metastasis, which can aid in the identification of novel prognostic genes and drug targets for treating the disease.

Short-Term Effects of Sepsis and the Impact of Aging on the Transcriptional Profile of Different Brain Regions.

Among the clinical manifestations observed in septic patients, sepsis-associated encephalopathy (SAE) is probably the most obscure and poorly explored. It is well established, however, that SAE is more prevalent in aged individuals and related to a worse outcome. In this context, we decided to investigate the acute effects of sepsis, induced by cecal ligation and puncture (CLP), on the cerebral transcriptional profile of young and old rats. The idea was to highlight important signaling pathways possibly implicated in the early stages of SAE. Global gene expression analysis of three different brain regions (hippocampus, cerebellum, and cortex) indicated a relatively small interference of sepsis at the transcriptional level. Cerebellum tissue was the least affected by sepsis in aged rats. The increased expression of S100a8, Upp1, and Mt2a in all three brain regions of young septic rats indicate that these genes may be involved in the first line of response to sepsis in the younger brain. On the other hand, altered expression of a network of genes involved in sensory perception of smell in the cortex of aged rats, but not in young ones, indicates an earlier disruption of cortex function, possibly more sensitive to the systemic inflammation. The expression of S100a8 at the protein level was confirmed in all brain regions, with clear-up regulation in septic aged cortex. Taken together, our results indicate that the transcriptional response of the central nervous system to early sepsis varies between distinct brain regions and that the cortex is affected earlier in aged animals, in line with early neurological manifestations observed in older patients.

Insights into the Function of Long Noncoding RNAs in Sepsis Revealed by Gene Co-Expression Network Analysis.

Sepsis is a major cause of death and its incidence and mortality increase exponentially with age. Most gene expression studies in sepsis have focused in protein-coding genes and the expression patterns, and potential roles of long noncoding RNAs (lncRNAs) have not been investigated yet. In this study, we performed co-expression network analysis of protein-coding and lncRNAs measured in neutrophil granulocytes from adult and elderly septic patients, along with age-matched healthy controls. We found that the genes displaying highest network similarity are predominantly differently expressed in sepsis and are enriched in loci encoding proteins with structural or regulatory functions related to protein translation and mitochondrial energetic metabolism. A number of lncRNAs are strongly connected to genes from these pathways and may take part in regulatory loops that are perturbed in sepsis. Among those, the ribosomal pseudogenes RP11-302F12.1 and RPL13AP7 are differentially expressed and appear to have a regulatory role on protein translation in both the elderly and adults, and lncRNAs MALAT1, LINC00355, MYCNOS, and AC010970.2 display variable connection strength and inverted expression patterns between adult and elderly networks, suggesting that they are the best candidates to be further studied to understand the mechanisms by which the immune response is impaired by age. In summary, we report the expression of lncRNAs that are deregulated in patients with sepsis, including subsets that display hub properties in molecular pathways relevant to the disease pathogenesis and that may participate in gene expression regulatory circuits related to the poorer disease outcome observed in elderly subjects.

Septic Shock in Advanced Age: Transcriptome Analysis Reveals Altered Molecular Signatures in Neutrophil Granulocytes.

Sepsis is one of the highest causes of mortality in hospitalized people and a common complication in both surgical and clinical patients admitted to hospital for non-infectious reasons. Sepsis is especially common in older people and its incidence is likely to increase substantially as a population ages. Despite its increased prevalence and mortality in older people, immune responses in the elderly during septic shock appear similar to that in younger patients. The purpose of this study was to conduct a genome-wide gene expression analysis of circulating neutrophils from old and young septic patients to better understand how aged individuals respond to severe infectious insult. We detected several genes whose expression could be used to differentiate immune responses of the elderly from those of young people, including genes related to oxidative phosphorylation, mitochondrial dysfunction and TGF-ß signaling, among others. Our results identify major molecular pathways that are particularly affected in the elderly during sepsis, which might have a pivotal role in worsening clinical outcomes compared with young people with sepsis.

Microarray gene expression analysis of neutrophils from elderly septic patients.

Sepsis is an especially common affliction in the elderly and despite its increased prevalence and mortality in older people, the immune response of the elderly during septic shock appears similar to that of younger patients. In the original study we conducted a global gene expression analysis of circulating neutrophils from elderly and young septic patients, as well as from age-matched healthy controls, to better understand how elder individuals respond to severe infectious insult (Pellegrina et al., 2015). Here we provide additional details pertaining processing and statistical analysis of the microarray data. Raw and normalized datasets linked to this project have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE67652.

A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system.

The aim of this study was to evaluate the anti-tumor activity of Amblyomin-X, a serine protease Kunitz-type inhibitor. Amblyomin-X induced tumor mass regression and decreased number of metastatic events in a B16F10 murine melanoma model. Alterations on expression of several genes related to cell cycle were observed when two tumor cell lines were treated with Amblyomin-X. PSMB2, which encodes a proteasome subunit, was differentially expressed, in agreement to inhibition of proteasomal activity in both cell lines. In conclusion, our results indicate that Amblyomin-X selectively acts on tumor cells by inducing apoptotic cell death, possibly by targeting the ubiquitin-proteasome system.