Genotoxicity and Gene Expression in the Rat Lung Tissue following Instillation and Inhalation of Different Variants of Amorphous Silica Nanomaterials (aSiO2 NM).

Several reports on amorphous silica nanomaterial (aSiO2 NM) toxicity have been questioning their safety. Herein, we investigated the in vivo pulmonary toxicity of four variants of aSiO2 NM: SiO2_15_Unmod, SiO2_15_Amino, SiO2_7 and SiO2_40. We focused on alterations in lung DNA and protein integrity, and gene expression following single intratracheal instillation in rats. Additionally, a short-term inhalation study (STIS) was carried out for SiO2_7, using TiO2_NM105 as a benchmark NM. In the instillation study, a significant but slight increase in oxidative DNA damage in rats exposed to the highest instilled dose (0.36 mg/rat) of SiO2_15_Amino was observed in the recovery (R) group. Exposure to SiO2_7 or SiO2_40 markedly increased oxidative DNA lesions in rat lung cells of the exposure (E) group at every tested dose. This damage seems to be repaired, since no changes compared to controls were observed in the R groups. In STIS, a significant increase in DNA strand breaks of the lung cells exposed to 0.5 mg/m3 of SiO2_7 or 50 mg/m3 of TiO2_NM105 was observed in both groups. The detected gene expression changes suggest that oxidative stress and/or inflammation pathways are likely implicated in the induction of (oxidative) DNA damage. Overall, all tested aSiO2 NM were not associated with marked in vivo toxicity following instillation or STIS. The genotoxicity findings for SiO2_7 from instillation and STIS are concordant; however, changes in STIS animals were more permanent/difficult to revert.

Modulation of Gene Expression in a Sterile Atopic Dermatitis Model and Inhibition of Staphylococcus aureus Adhesion by Fucoidan.

Atopic dermatitis is a multifactorial pathology that includes perturbations of gene expression and increased adhesion of Staphylococcus aureus. Fucoidans are seaweed-derived sulfated fucose-rich polysaccharides that are known to be anti-inflammatory and may inhibit adhesion of pathogens. Fucoidan was assessed for effects on gene expression of an in vitro 3D model of atopic dermatitis. It was also assessed for inhibitory effects on the adhesion of bacteria onto 3D reconstructed skin. Fucoidan significantly altered gene expression in the atopic dermatitis model, and there was a trend to reduce periostin levels. Fucoidan significantly inhibited the adhesion of Staphylococcus aureus and Cutibacterium acnes but did not affect the adhesion of Staphylococcus epidermidis. Fucoidan may be a useful topical agent to assist in the management of atopic dermatitis.

On the relevance of an in vitro reconstructed human epidermis model for drug screening in atopic dermatitis.

Recent advances in the development of human-based in vitro models offer new tools for drug screening and mechanistic investigations of new therapeutic agents. However, there is a lack of evidence that disease models respond favourably to potential drug candidates. Atopic dermatitis (AD) is a very common disease associated with an altered skin barrier and chronic inflammation. Here, we demonstrate that the AD-like features of a reconstructed human epidermis (RHE) model treated with Th2 cytokines are reversed in the presence of molecules known to have a beneficial effect on damaged skin as a result of modulating various signalling cascades including the Liver X Receptors and JAK/STAT pathways. This work shows that standardized and reproducible RHE are relevant models for therapeutic research assessing new drug candidates aiming to restore epidermal integrity in an inflammatory environment.

Genomics and Epigenetics of Malignant Mesothelioma.

Malignant mesothelioma is an aggressive and lethal asbestos-related disease. Diagnosis of malignant mesothelioma is particularly challenging and is further complicated by the lack of disease subtype-specific markers. As a result, it is especially difficult to distinguish malignant mesothelioma from benign reactive mesothelial proliferations or reactive fibrosis. Additionally, mesothelioma diagnoses can be confounded by other anatomically related tumors that can invade the pleural or peritoneal cavities, collectively resulting in delayed diagnoses and greatly affecting patient management. High-throughput analyses have uncovered key genomic and epigenomic alterations driving malignant mesothelioma. These molecular features have the potential to better our understanding of malignant mesothelioma biology as well as to improve disease diagnosis and patient prognosis. Genomic approaches have been instrumental in identifying molecular events frequently occurring in mesothelioma. As such, we review the discoveries made using high-throughput technologies, including novel insights obtained from the analysis of the non-coding transcriptome, and the clinical potential of these genetic and epigenetic findings in mesothelioma. Furthermore, we aim to highlight the potential of these technologies in the future clinical applications of the novel molecular features in malignant mesothelioma.

Breast cancer associated germline structural variants harboring small noncoding RNAs impact post-transcriptional gene regulation.

Copy Number Variants (CNVs) are a class of structural variations of DNA. Germline CNVs are known to confer disease susceptibility, but their role in breast cancer warrants further investigations. We hypothesized that breast cancer associated germline CNVs contribute to disease risk through gene dosage or other post-transcriptional regulatory mechanisms, possibly through tissue specific expression of CNV-embedded small-noncoding RNAs (CNV-sncRNAs). Our objectives are to identify breast cancer associated CNVs using a genome wide association study (GWAS), identify sncRNA genes embedded within CNVs, confirm breast tissue (tumor and normal) expression of the sncRNAs, correlate their expression with germline copy status and identify pathways influenced by the genes regulated by sncRNAs. We used an association study design and accessed germline CNV data generated on Affymetrix Human SNP 6.0 array in 686 (in-house data) and 495 (TCGA data) subjects served as discovery and validation cohorts. We identified 1812 breast cancer associated CNVs harboring miRNAs (n = 38), piRNAs (n = 9865), snoRNAs (n = 71) and tRNAs (n = 12) genes. A subset of CNV-sncRNAs expressed in breast tissue, also showed correlation with germline copy status. We identified targets potentially regulated by miRNAs and snoRNAs. In summary, we demonstrate the potential impact of embedded CNV-sncRNAs on expression and regulation of down-stream targets.

Environmental arsenic exposure: From genetic susceptibility to pathogenesis.

More than 200 million people in 70 countries are exposed to arsenic through drinking water. Chronic exposure to this metalloid has been associated with the onset of many diseases, including cancer. Epidemiological evidence supports its carcinogenic potential, however, detailed molecular mechanisms remain to be elucidated. Despite the global magnitude of this problem, not all individuals face the same risk. Susceptibility to the toxic effects of arsenic is influenced by alterations in genes involved in arsenic metabolism, as well as biological factors, such as age, gender and nutrition. Moreover, chronic arsenic exposure results in several genotoxic and epigenetic alterations tightly associated with the arsenic biotransformation process, resulting in an increased cancer risk. In this review, we: 1) review the roles of inter-individual DNA-level variations influencing the susceptibility to arsenic-induced carcinogenesis; 2) discuss the contribution of arsenic biotransformation to cancer initiation; 3) provide insights into emerging research areas and the challenges in the field; and 4) compile a resource of publicly available arsenic-related DNA-level variations, transcriptome and methylation data. Understanding the molecular mechanisms of arsenic exposure and its subsequent health effects will support efforts to reduce the worldwide health burden and encourage the development of strategies for managing arsenic-related diseases in the era of personalized medicine.

Germline copy number variations are associated with breast cancer risk and prognosis.

Breast cancer is one of the most common cancers among women, and susceptibility is explained by genetic, lifestyle and environmental components. Copy Number Variants (CNVs) are structural DNA variations that contribute to diverse phenotypes via gene-dosage effects or cis-regulation. In this study, we aimed to identify germline CNVs associated with breast cancer susceptibility and their relevance to prognosis. We performed whole genome CNV genotyping in 422 cases and 348 controls using Human Affymetrix SNP 6 array. Principal component analysis for population stratification revealed 84 outliers leaving 366 cases and 320 controls of Caucasian ancestry for association analysis; CNVs with frequency > 10% and overlapping with protein coding genes were considered for breast cancer risk and prognostic relevance. Coding genes within the CNVs identified were interrogated for gene- dosage effects by correlating copy number status with gene expression profiles in breast tumor tissue. We identified 200 CNVs associated with breast cancer (q-value < 0.05). Of these, 21 CNV regions (overlapping with 22 genes) also showed association with prognosis. We validated representative CNVs overlapping with APOBEC3B and GSTM1 genes using the TaqMan assay. Germline CNVs conferred dosage effects on gene expression in breast tissue. The candidate CNVs identified in this study warrant independent replication.

Gene expression analysis of microtubule affinity-regulating kinase 2 in non-small cell lung cancer.

Lung cancer is the leading cause of cancer death worldwide, and has a five-year survival rate of 18% [1]. MARK2 is a serine/threonine-protein kinase, and is a key component in the phosphorylation of microtubule-associated proteins [2], [3]. A recent study published by Hubaux et al. found that microtubule affinity-regulating kinase 2 (MARK2) showed highly frequent DNA and RNA level disruption in lung cancer cell lines and independent non-small cell lung cancer (NSCLC) cohorts [4]. These alterations result in the acquisition of oncogenic properties in cell lines, such as increased viability and anchorage-independent growth. Furthermore, a microarray-based transcriptome analysis of three short hairpin RNA (shRNA)-mediated MARK2 knockdown lung adenocarcinoma cell lines (GEO#: GSE57966) revealed an association between MARK2 gene expression and cell cycle activation and DNA damage response. Here, we present a detailed description of transcriptome analysis to support the described role of MARK2 in promoting a malignant phenotype.

Unique somatic and malignant expression patterns implicate PIWI-interacting RNAs in cancer-type specific biology.

Human PIWI-interacting RNAs (piRNAs) are known to be expressed in germline cells, functionally silencing LINEs and SINEs. Their expression patterns in somatic tissues are largely uncharted. We analyzed 6,260 human piRNA transcriptomes derived from non-malignant and tumour tissues from 11 organs. We discovered that only 273 of the 20,831 known piRNAs are expressed in somatic non-malignant tissues. However, expression patterns of these piRNAs were able to distinguish tissue-of-origin. A total of 522 piRNAs are expressed in corresponding tumour tissues, largely distinguishing tumour from non-malignant tissues in a cancer-type specific manner. Most expressed piRNAs mapped to known transcripts, contrary to "piRNA clusters" reported in germline cells. We showed that piRNA expression can delineate clinical features, such as histological subgroups, disease stages, and survival. PiRNAs common to many cancer types might represent a core gene-set that facilitates cancer growth, while piRNAs unique to individual cancer types likely contribute to cancer-specific biology.

Microtubule affinity-regulating kinase 2 is associated with DNA damage response and cisplatin resistance in non-small cell lung cancer.

Microtubule affinity-regulating kinases (MARKs) are involved in several cellular functions but few studies have correlated MARK kinase expression with cancer, and none have explored their role in lung cancer. In this study, we identified MARK2 as frequently disrupted by DNA hypomethylation and copy gain, resulting in concordant overexpression in independent lung tumor cohorts and we demonstrate a role for MARK2 in lung tumor biology. Manipulation of MARK2 in lung cell lines revealed its involvement in cell viability and anchorage-independent growth. Analyses of both manipulated cell lines and clinical tumor specimens identified a potential role for MARK2 in cell cycle activation and DNA repair. Associations between MARK2 and the E2F, Myc/Max and NF-κB pathways were identified by luciferase assays and in-depth assessment of the NF-κB pathway suggests a negative association between MARK2 expression and NF-κB due to activation of non-canonical NF-κB signaling. Finally, we show that high MARK2 expression levels correlate with resistance to cisplatin, a standard first line chemotherapy for lung cancer. Collectively, our work supports a role for MARK2 in promoting malignant phenotypes of lung cancer and potentially modulating response to the DNA damaging chemotherapeutic, cisplatin.

Valproic acid improves second-line regimen of small cell lung carcinoma in preclinical models.

With 5-year survival rates below 5%, small cell lung carcinoma (SCLC) has very poor prognosis and requires improved therapies. Despite an excellent overall response to first-line therapy, relapses are frequent and further treatments are disappointing. The goal of the study was to improve second-line therapy of SCLC. The effect of chemotherapeutic agents was evaluated in cell lines (apoptosis, reactive oxygen species, and RNA and protein expression) and in mouse models (tumour development). We demonstrate here that valproic acid, a histone deacetylase inhibitor, improves the efficacy of a second-line regimen (vindesine, doxorubicin and cyclophosphamide) in SCLC cells and in mouse models. Transcriptomic profiling integrating microRNA and mRNA data identifies key signalling pathways in the response of SCLC cells to valproic acid, opening new prospects for improved therapies.

Unique pattern of component gene disruption in the NRF2 inhibitor KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex in serous ovarian cancer.

The NFE2-related factor 2 (NRF2) pathway is critical to initiate responses to oxidative stress; however, constitutive activation occurs in different cancer types, including serous ovarian carcinomas (OVCA). The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is a regulator of NRF2 levels. Hence, we investigated the DNA-level mechanisms affecting these genes in OVCA. DNA copy-number loss (CNL), promoter hypermethylation, mRNA expression, and sequence mutation for KEAP1, CUL3, and RBX1 were assessed in a cohort of 568 OVCA from The Cancer Genome Atlas. Almost 90% of cases exhibited loss-of-function alterations in any components of the NRF2 inhibitory complex. CNL is the most prominent mechanism of component disruption, with RBX1 being the most frequently disrupted component. These alterations were associated with reduced mRNA expression of complex components, and NRF2 target gene expression was positively enriched in 90% of samples harboring altered complex components. Disruption occurs through a unique DNA-level alteration pattern in OVCA. We conclude that a remarkably high frequency of DNA and mRNA alterations affects components of the KEAP1/CUL3/RBX1 complex, through a unique pattern of genetic mechanisms. Together, these results suggest a key role for the KEAP1/CUL3/RBX1 complex and NRF2 pathway deregulation in OVCA.

Whole-genome sequencing analysis identifies a distinctive mutational spectrum in an arsenic-related lung tumor.

INTRODUCTION: Arsenic exposure is a significant cause of lung cancer in North America and worldwide. Arsenic-related tumors are structurally indistinguishable from those induced by other carcinogens. Because carcinogens, like tobacco, induce distinctive mutational signatures, we sought to characterize the mutational signature of an arsenic-related lung tumor from a never smoker with the use of whole-genome sequencing. METHODS: Tumor and lung tissues were obtained from a never smoker with lung squamous cell carcinoma (LUSC), without familiar history of lung cancer and chronically exposed to high levels of arsenic-contaminated drinking water. The Illumina HiSeq-2000 platform was used to sequence each genome at approximately 30-fold haploid coverage. The mutational signature was compared with those observed in previously characterized lung tumors. RESULTS: The arsenic-related tumor exhibited alterations common in LUSC, such as the increased number of copies at 3q26 (SOX2 locus). However, the arsenic-related genome not only harbored a lower number of point mutations, but also had a remarkably high fraction of T>G/A>C mutations and low fraction of C>A/G>T transversions, which is uncharacteristic of LUSCs. Furthermore, at the gene level, we identified a rare G>C mutation in TP53, which is uncommon in lung tumors in general (<0.2%) but has been observed in other arsenic-related malignancies. CONCLUSIONS: We generated the first whole-genome sequence of an LUSC from a never-smoker patient chronically exposed to arsenic, and identified a distinct mutational spectrum associated with arsenic exposure, providing novel evidence supporting the hypothesis that arsenic-induced lung tumors arise through molecular mechanisms that differ from those of the common lung cancer.

Frequent concerted genetic mechanisms disrupt multiple components of the NRF2 inhibitor KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex in thyroid cancer.

BACKGROUND: Reactive oxygen species contribute to normal thyroid function. The NRF2 oxidative response pathway is frequently and constitutively activated in multiple tumor types, including papillary thyroid carcinoma (PTC). Genetic mechanisms underlying NRF2 pathway activation in PTC are not fully understood. Thus, we aimed to determine whether inactivating patterns of DNA-level alterations affect genes encoding for individual NRF2 inhibitor complex components (CUL3/KEAP1/RBX1) occur in PTC. FINDINGS: Combined patterns of epi/genetic alterations for KEAP1/CUL3/RBX1 E3 ubiquitin-ligase complex components were simultaneously interrogated for a panel of 310 PTC cases and 40 adjacent non-malignant tissues. Data were obtained from The Cancer Genome Atlas project. Enrichment of NRF2 pathway activation was assessed by gene-set enrichment analysis using transcriptome data. Our analyses revealed that PTC sustain a strikingly high frequency (80.6%) of disruption to multiple component genes of the NRF2 inhibitor complex. Hypermethylation is the predominant inactivating mechanism primarily affecting KEAP1 (70.6%) and CUL3 (20%), while copy number loss mostly affects RBX1 (16.8%). Concordantly, NRF2-associated gene expression signatures are positively and significantly enriched in PTC. CONCLUSIONS: The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is almost ubiquitously affected by multiple DNA-level mechanisms and downstream NRF2 pathway targets are activated in PTC. Given the importance of this pathway to normal thyroid function as well as to cancer; targeted inhibition of NRF2 regulators may impact strategies for therapeutic intervention involving this pathway.

EZH2 promotes E2F-driven SCLC tumorigenesis through modulation of apoptosis and cell-cycle regulation.

INTRODUCTION: Although enhancer of zeste homolog 2 (EZH2) has been associated with both non-small cell and small-cell lung cancers (SCLCs), current observations suggest different mechanisms of EZH2 activation and overexpression in these lung cancer types. Globally, SCLC kills 200,000 people yearly. New clinical approaches for SCLC treatment are required to improve the poor survival rate. Given the therapeutic potential of EZH2 as a target, we sought to delineate the downstream consequences of EZH2 disruption to identify the cellular mechanisms by which EZH2 promotes tumorigenesis in SCLC. METHODS: We generated cells with stable expression of short hairpin RNA targeting EZH2 and corresponding controls (pLKO.1) and determined the consequences of EZH2 knockdown on the cell cycle and apoptosis by means of propidium iodide staining and fluorescence-activated cell sorting, Western blot, quantitative reverse transcriptase-polymerase chain reaction as well as cell viability assessment using methylthiazol tetrazolium assays. RESULTS: We discovered that EZH2 inhibition (1) increased apoptotic activity by up-regulating the proapoptotic factors Puma and Bad, (2) decreased the fraction of cells in S or G2/M phases, and (3) elevated p21 protein levels, implicating EZH2 in cell death and cell-cycle control in SCLC. CONCLUSION: Our findings present evidence for the role of EZH2 in the regulation of cell cycle and apoptosis, providing a biological mechanism to explain the tumorigenicity of EZH2 in SCLC. Our work points to the great potential of EZH2 as a therapeutic target in SCLC.

The role of epigenetics in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an almost invariably fatal cancer of the pleura due to asbestos exposure. Increasing evidence indicates that unresponsiveness to chemotherapy is due to epigenetic errors leading to inadequate gene expression in tumor cells. The availability of compounds that modulate epigenetic modifications, such as histone acetylation or DNA methylation, offers new prospects for treatment of MPM. Here, we review latest findings on epigenetics in mesothelioma and present novel strategies for promising epigenetic therapies.

Molecular features in arsenic-induced lung tumors.

Arsenic is a well-known human carcinogen, which potentially affects ~160 million people worldwide via exposure to unsafe levels in drinking water. Lungs are one of the main target organs for arsenic-related carcinogenesis. These tumors exhibit particular features, such as squamous cell-type specificity and high incidence among never smokers. Arsenic-induced malignant transformation is mainly related to the biotransformation process intended for the metabolic clearing of the carcinogen, which results in specific genetic and epigenetic alterations that ultimately affect key pathways in lung carcinogenesis. Based on this, lung tumors induced by arsenic exposure could be considered an additional subtype of lung cancer, especially in the case of never-smokers, where arsenic is a known etiological agent. In this article, we review the current knowledge on the various mechanisms of arsenic carcinogenicity and the specific roles of this metalloid in signaling pathways leading to lung cancer.

Arsenic, asbestos and radon: emerging players in lung tumorigenesis.

The cause of lung cancer is generally attributed to tobacco smoking. However lung cancer in never smokers accounts for 10 to 25% of all lung cancer cases. Arsenic, asbestos and radon are three prominent non-tobacco carcinogens strongly associated with lung cancer. Exposure to these agents can lead to genetic and epigenetic alterations in tumor genomes, impacting genes and pathways involved in lung cancer development. Moreover, these agents not only exhibit unique mechanisms in causing genomic alterations, but also exert deleterious effects through common mechanisms, such as oxidative stress, commonly associated with carcinogenesis. This article provides a comprehensive review of arsenic, asbestos, and radon induced molecular mechanisms responsible for the generation of genetic and epigenetic alterations in lung cancer. A better understanding of the mode of action of these carcinogens will facilitate the prevention and management of lung cancer related to such environmental hazards.