Metagenomic characterization of parental and production CHO cell lines for detection of adventitious viruses.

Viral contamination is a major concern for biological products. Therefore, virus testing of raw materials and cells is essential for the safety of the final product. We used high-throughput sequencing to detect viral-like sequences in selected CHO cell lines. Our aim was to test various approaches of sample preparation, to establish a pipeline for metagenomic analysis and to characterize standard viral metagenome of production and parental CHO cell lines. The comparison of the metagenomics composition of the differently prepared samples showed that among four tested approaches sequencing of ribosomal RNA depleted total RNA is the most promising approach. The metagenomics investigation of one production and three parental CHO cell lines of diverse origin did not indicate the presence of adventitious viral agents in the investigated samples. The study revealed an expected background of virus-like nucleic acids in the samples, which originate from remains of expression vectors, endogenized viral elements and residuals of bacteriophages.

Genetic associations of the interleukin locus at 1q32.1 with clinical outcomes of cutaneous melanoma.

BACKGROUND: Due to high melanoma immunogenicity, germline genetic variants in immune pathways have been studied for association with melanoma prognosis. However, limited candidate selection, inadequate power, or lack of independent validation have hampered the reproducibility of these prior findings, preventing personalised clinical applicability in melanoma prognostication. Our objective was to assess the prognostic utility of genetic variants in immunomodulatory pathways for prediction of melanoma clinical outcomes. METHODS: We genotyped 72 tag single nucleotide polymorphisms (SNPs) in 44 immunomodulatory genes in a population sample of 1022 melanoma patients and performed Cox regression analysis to test the association between SNPs and melanoma recurrence-free (RFS) and overall survival (OS). We have further investigated the most significant associations using a fine mapping strategy and followed with functional analyses in CD4+ T cells in a subset of 75 melanoma patients. RESULTS: The most significant associations were found with melanoma OS for rs3024493 in IL10 at chromosome 1q32.1 (heterozygous HR 0.58, 95% CI 0.39 to 0.86; p=0.0006), a variant previously shown to be linked with autoimmune conditions. Multiple additional SNPs at 1q32.1 were also nominally associated with OS confirming at least two independent association signals in this locus. In addition, we found rs3024493 associated with the downregulation of interleukin 10 (IL10) secretion in CD4+ T cells. CONCLUSIONS: We discovered novel associations of IL10 with melanoma survival at 1q32.1, suggesting this locus should be considered as a novel melanoma prognostic biomarker with potential for aiding melanoma patient management. Our findings also provide further support for an alternative role of IL10 in stimulation of anti-tumour immune response.

NAT1 and NAT2 genetic polymorphisms and environmental exposure as risk factors for oesophageal squamous cell carcinoma: a case-control study.

BACKGROUND: Tobacco smoking and red meat consumption are some of the known risk factors associated with the development of oesophageal cancer. N-acetytransferases (NAT1 and NAT2) play a key role in metabolism of carcinogenic arylamines present in tobacco smoke and overcooked red meat. We hypothesized that NAT1 and NAT2 genetic polymorphisms may influence the risk of oesophageal cancer upon exposure to environmental carcinogens. METHODS: Single nucleotide polymorphisms (SNPs) in the NAT1 and NAT2 genes were investigated by genotyping 732 cases and 768 healthy individuals from two South African populations to deduce the acetylator phenotype (slow, intermediate or rapid) from the combination of the genotyped SNPs. RESULTS: The 341 CC genotype (rs1801280) was significantly associated with a reduced risk for oesophageal cancer in the Mixed Ancestry population (OR = 0.31; 95% CI 0.11-0.87). The NAT2 slow/intermediate acetylator status significantly increased the risk among cigarette smokers in the Black population (OR = 2.76; 95% CI 1.69-4.52), as well as among alcohol drinkers in the Mixed Ancestry population (OR = 2.77; 95% CI 1.38-5.58). Similarly, the NAT1 slow/intermediate acetylator status was a risk factor for tobacco smokers in the Black population (OR = 3.41; 95% CI 1.95-5.96) and for alcohol drinkers in the Mixed Ancestry population (OR = 3.41; 95% CI 1.70-6.81). In a case-only analysis, frequent red meat consumption was associated with a significantly increased cancer risk for NAT2 slow/intermediate acetylators in the Mixed Ancestry population (OR = 3.55; 95% CI 1.29-9.82; P = 0.019), whereas daily white meat intake was associated with an increased risk among NAT1 slow/intermediate acetylators in the Black population (OR = 1.82; 95% CI 1.09-3.04; P = 0.023). CONCLUSIONS: Our findings indicate that N-acetylation polymorphisms may modify the association between environmental risk factors and oesophageal cancer risk and that N-acetyltransferases may play a key role in detoxification of carcinogens. Prevention strategies in lifestyle and dietary habits may reduce the incidence of oesophageal cancer in high-risk populations.

The receptor tyrosine kinase Axl in cancer: biological functions and therapeutic implications.

The receptor tyrosine kinase Axl has been implicated in the malignancy of different types of cancer. Emerging evidence of Axl upregulation in numerous cancers, as well as reports demonstrating that its inhibition blocks tumor formation in animal models, highlight the importance of Axl as a new potential therapeutic target. Furthermore, recent data demonstrate that Axl plays a pivotal role in resistance to chemotherapeutic regimens. In this review we discuss the functions of Axl and its regulation and role in cancer development, resistance to therapy, and its importance as a potential drug target, focusing on acute myeloid leukemia, breast, prostate and non-small cell lung cancers.

The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification.

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.

Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast.

BACKGROUND: Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells. METHODS: Yeast MLH1 and PMS1 genes, whose protein products form the MutLalpha complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested. RESULTS: The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic. CONCLUSION: Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.

Germline determinants of clinical outcome of cutaneous melanoma.

Cutaneous melanoma (CM) is the most lethal form of skin cancer. Despite the constant increase in melanoma incidence, which is in part due to incremental advances in early diagnostic modalities, mortality rates have not improved over the last decade and for advanced stages remain steadily high. While conventional prognostic biomarkers currently in use find significant utility for predicting overall general survival probabilities, they are not sensitive enough for a more personalized clinical assessment on an individual level. In recent years, the advent of genomic technologies has brought the promise of identification of germline DNA alterations that may associate with CM outcomes and hence represent novel biomarkers for clinical utilization. This review attempts to summarize the current state of knowledge of germline genetic factors studied for their impact on melanoma clinical outcomes. We also discuss ongoing problems and hurdles in validating such surrogates, and we also project future directions in discovery of more powerful germline genetic factors with clinical utility in melanoma prognostication.

Wharton's Jelly-Derived Mesenchymal Stromal Cells and Fibroblast-Derived Extracellular Matrix Synergistically Activate Apoptosis in a p21-Dependent Mechanism in WHCO1 and MDA MB 231 Cancer Cells In Vitro.

The tumour microenvironment plays a crucial role in tumour progression and comprises tumour stroma which is made up of different cell types and the extracellular matrix (ECM). Mesenchymal stromal cells (MSCs) are part of the tumour stroma and may have conflicting effects on tumour growth. In this study we investigated the effect of Wharton's Jelly-derived MSCs (WJ-MSCs) and a fibroblast-derived ECM (fd-ECM) on esophageal (WHCO1) and breast (MDA MB 231) cancer cells in vitro. Both WJ-MSCs and the fd-ECM, alone or in combination, downregulate PCNA, cyclin D1, Bcl-2, Bcl-xL, and MMPs and upregulate p53 and p21. p21 induction resulted in G2 phase cell cycle arrest and induced apoptosis in vitro. Our data suggest that p21 induction is via p53-dependent and p53-independent mechanisms in WHCO1 and MDA MB 231 cells, respectively. Vascular endothelial growth factor, Akt, and Nodal pathways were downregulated in cancer cells cocultured with WJ-MSCs. We also demonstrate that WJ-MSCs effects on cancer cells appear to be short-lived whilst the fd-ECM effect is long-lived. This study shows the influence of tumour microenvironment on cancer cell behaviour and provides alternative therapeutic targets for potential regulation of tumour cells.

The Expression Quantitative Trait Loci in Immune Pathways and their Effect on Cutaneous Melanoma Prognosis.

PURPOSE: The identification of personalized germline markers with biologic relevance for the prediction of cutaneous melanoma prognosis is highly demanded but to date, it has been largely unsuccessful. As melanoma progression is controlled by host immunity, here we present a novel approach interrogating immunoregulatory pathways using the genome-wide maps of expression quantitative trait loci (eQTL) to reveal biologically relevant germline variants modulating cutaneous melanoma outcomes. EXPERIMENTAL DESIGN: Using whole genome eQTL data from a healthy population, we identified 385 variants significantly impacting the expression of 268 immune-relevant genes. The 40 most significant eQTLs were tested in a prospective cohort of 1,221 patients with cutaneous melanoma for their association with overall (OS) and recurrence-free survival using Cox regression models. RESULTS: We identified highly significant associations with better melanoma OS for rs6673928, impacting IL19 expression (HR, 0.56; 95% CI, 0.41-0.77; P = 0.0002) and rs6695772, controlling the expression of BATF3 (HR, 1.64; 95% CI, 1.19-2.24; P = 0.0019). Both associations map in the previously suspected melanoma prognostic locus at 1q32. Furthermore, we show that their combined effect on melanoma OS is substantially enhanced reaching the level of clinical applicability (HR, 1.92; 95% CI, 1.43-2.60; P = 2.38e-5). CONCLUSIONS: Our unique approach of interrogating lymphocyte-specific eQTLs reveals novel and biologically relevant immunomodulatory eQTL predictors of cutaneous melanoma prognosis that are independent of current histopathologic markers. The significantly enhanced combined effect of identified eQTLs suggests the personalized utilization of both SNPs in a clinical setting, strongly indicating the promise of the proposed design for the discovery of prognostic or risk germline markers in other cancers. Clin Cancer Res; 22(13); 3268-80. ©2016 AACR.

Identification of a Novel Pathogenic Germline KDR Variant in Melanoma.

PURPOSE: The application of pan-cancer next-generation sequencing panels in the clinical setting has facilitated the identification of low frequency somatic mutations and the testing of new therapies in solid tumors using the "basket trial" scheme. However, little consideration has been given to the relevance of nonsynonymous germline variants, which are likely to be uncovered in tumors and germline and which may be relevant to prognostication and prediction of treatment response. EXPERIMENTAL DESIGN: We analyzed matched tumor and normal DNA from 34 melanoma patients using an Ion Torrent cancer-associated gene panel. We elected to study the germline variant Q472H in the kinase insert domain receptor (KDR), which was identified in 35% of melanoma patients in both a pilot and an independent 1,223 patient cohort. Using patient-derived melanoma cell lines and human samples, we assessed proliferation, invasion, VEGF levels, and angiogenesis by analyzing tumor microvessel density (MVD) using anti-CD34 antibody. RESULTS: Serum VEGF levels and tumor MVD were significantly higher in Q472H versus KDR wild-type (WD) patients. Primary cultures derived from melanomas harboring the KDR variant were more proliferative and invasive than KDR wild type. Finally, using a VEGFR2 antibody, we showed that KDR Q472H cells were sensitive to targeted inhibition of VEGFR2, an effect that was not observed in KDR WT cells. CONCLUSIONS: Our data support the integration of germline analysis into personalized treatment decision-making and suggest that patients with germline KDR variant might benefit from antiangiogenesis treatment. Clin Cancer Res; 22(10); 2377-85. ©2015 AACR.

Genetic markers of pigmentation are novel risk loci for uveal melanoma.

While the role of genetic risk factors in the etiology of uveal melanoma (UM) has been strongly suggested, the genetic susceptibility to UM is currently vastly unexplored. Due to shared epidemiological risk factors between cutaneous melanoma (CM) and UM, in this study we have selected 28 SNPs identified as risk variants in previous genome-wide association studies on CM or CM-related host phenotypes (such as pigmentation and eye color) and tested them for association with UM risk. By logistic regression analysis of 272 UM cases and 1782 controls using an additive model, we identified five variants significantly associated with UM risk, all passing adjustment for multiple testing. The three most significantly associated variants rs12913832 (OR = 0.529, 95% CI 0.415-0.673; p = 8.47E-08), rs1129038 (OR = 0.533, 95% CI 0.419-0.678; p = 1.19E-07) and rs916977 (OR = 0.465, 95% CI 0.339-0.637; p = 3.04E-07) are correlated (r(2) > 0.5) and map at 15q12 in the region of HERC2/OCA2, which determines eye-color in the human population. Our data provides first evidence that the genetic factors associated with pigmentation traits are risk loci of UM susceptibility.

Wnt/ß-Catenin and MEK-ERK Signaling are Required for Fibroblast-Derived Extracellular Matrix-Mediated Endoderm Differentiation of Embryonic Stem Cells.

Human embryonic stem cells (hESCs) have the potential to differentiate into all cells of the three germ layers, thus making them an attractive source of cells for use in regenerative medicine. The greatest challenge lies in regulating the differentiation of hESCs into specific cell lineages by both intrinsic and extrinsic factors. In this study we determined the effect of a fibroblast-derived extracellular matrix (fd-ECM) on hESCs differentiation. We demonstrate that growth of hESCs on fd-ECM results in hESCs losing their stemness and proliferation potential. As the stem cells differentiate they attain gene expression profiles similar to the primitive streak of the in vivo embryo. The activation of both the MEK-ERK and Wnt/ß-catenin signaling pathways is required for the fd-ECM-mediated differentiation of hESCs towards the endoderm and involves integrins α1, α2, α3 and ß1. This study illustrates the importance of the cellular microenvironment in directing stem cell fate and that the nature and composition of the extracellular matrix is a crucial determining factor.

Aberrant methylation of the MSH3 promoter and distal enhancer in esophageal cancer patients exposed to first-hand tobacco smoke.

PURPOSE: Polymorphisms in MSH3 gene confer risk of esophageal cancer when in combination with tobacco smoke exposure. The purpose of this study was to investigate the methylation status of MSH3 gene in esophageal cancer patients in order to further elucidate possible role of MSH3 in esophageal tumorigenesis. METHODS: We applied nested methylation-specific polymerase chain reaction to investigate the methylation status of the MSH3 promoter in tumors and matching adjacent normal-looking tissues of 84 esophageal cancer patients from a high-risk South African population. The Cancer Genome Atlas data were used to examine DNA methylation profiles at 17 CpG sites located in the MSH3 locus. RESULTS: Overall, promoter methylation was detected in 91.9 % of tumors, which was significantly higher compared to 76.0 % in adjacent normal-looking esophageal tissues (P = 0.008). When samples were grouped according to different demographics (including age, gender and ethnicity) and smoking status of patients, methylation frequencies were found to be significantly higher in tumor tissues of Black subjects (P = 0.024), patients of 55-65 years of age (P = 0.032), males (P = 0.037) and tobacco smokers (P = 0.015). Furthermore, methylation of the MSH3 promoter was significantly more frequent in tumor samples from smokers compared to tumor samples from non-smokers [odds ratio (OR) = 31.9, P = 0.031]. The TCGA data confirmed significantly higher DNA methylation level at the MSH3 promoter region in tumors (P = 0.0024). In addition, we found evidence of an aberrantly methylated putative MSH3-associated distal enhancer element. CONCLUSION: Our results suggest that methylation of MSH3 together with exposure to tobacco smoke is involved in esophageal carcinogenesis. Due to the active role of the MSH3 protein in modulating chemosensitivity of cells, methylation of MSH3 should further be examined in association with the outcome of esophageal cancer treatment using anticancer drugs.

MicroRNA polymorphisms and environmental smoke exposure as risk factors for oesophageal squamous cell carcinoma.

MicroRNAs (miRNAs) and related polymorphisms have been implicated in the susceptibility to oesophageal squamous cell carcinoma (OSCC). In our study, three miRNA-related SNPs: rs6505162 A>C (pre-miRNA of miR-423), rs213210 A>G (3'UTR of miR-219-1) and rs7372209 C>T (5'UTR of miR-26a-1) were investigated in the Black and Mixed Ancestry population groups in South Africa. The potential cumulative effects of these SNPs, as well as gene-environment interactions were also analysed. In Blacks, rs6505162 A>C was associated with OSCC under dominant, additive and recessive models with odds ratios (ORs) 1.353, 1.404, and 2.858, respectively. This locus showed very strong interactions with smoke inhalation from burning wood or charcoal used for heating and cooking in very poorly ventilated areas (OR(GE)=7.855, P(GE)=9.17*10(-10) in the Black group). Furthermore, the miR-423-3p level was 1.39 fold up-regulated in tumour tissues compared to the adjacent normal tissue (paired t-test P value 0.0087). SNP-SNP interaction between rs2132210 and rs7372209 was found in both Black and Mixed Ancestry subjects. The AArs213210-CTrs7372209 genotype had a protective effect on OSCC risk (in the Black, OR=0.229, P=0.012; and the Mixed Ancestry groups, OR=0.230, P=0.00014). This study is the first to link SNPs in miR-423 together with environmental smoke exposure to risk for developing OSCC.

The cumulative effects of polymorphisms in the DNA mismatch repair genes and tobacco smoking in oesophageal cancer risk.

The DNA mismatch repair (MMR) enzymes repair errors in DNA that occur during normal DNA metabolism or are induced by certain cancer-contributing exposures. We assessed the association between 10 single-nucleotide polymorphisms (SNPs) in 5 MMR genes and oesophageal cancer risk in South Africans. Prior to genotyping, SNPs were selected from the HapMap database, based on their significantly different genotypic distributions between European ancestry populations and four HapMap populations of African origin. In the Mixed Ancestry group, the MSH3 rs26279 G/G versus A/A or A/G genotype was positively associated with cancer (OR = 2.71; 95% CI: 1.34-5.50). Similar associations were observed for PMS1 rs5742938 (GG versus AA or AG: OR = 1.73; 95% CI: 1.07-2.79) and MLH3 rs28756991 (AA or GA versus GG: OR = 2.07; 95% IC: 1.04-4.12). In Black individuals, however, no association between MMR polymorhisms and cancer risk was observed in individual SNP analysis. The interactions between MMR genes were evaluated using the model-based multifactor-dimensionality reduction approach, which showed a significant genetic interaction between SNPs in MSH2, MSH3 and PMS1 genes in Black and Mixed Ancestry subjects, respectively. The data also implies that pathogenesis of common polymorphisms in MMR genes is influenced by exposure to tobacco smoke. In conclusion, our findings suggest that common polymorphisms in MMR genes and/or their combined effects might be involved in the aetiology of oesophageal cancer.

Non-truncating hMLH1 variants identified in Slovenian gastric cancer patients are not associated with Lynch Syndrome: a functional analysis report.

Hereditary non-polyposis colorectal cancer is the most common known genetic syndrome that predisposes to various types of cancer including gastric cancer and occures mainly due to pathogenic germline mutations in DNA mismatch repair (MMR) genes, such as MLH1, MSH2 and MSH6. Impaired MMR activity can lead to microsatellite instability (MSI) in tumor tissues. Interpreting the pathogenic significance of identified mutations in MMR genes, especially missense alterations and short in-frame deletions and insertions is challenging and functional analysis is often needed to accurately assess their pathogenicities. The purpose of this study was to evaluate functional significance of MLH1 missense mutations, previously identified in unrelated Slovenian patients with MSI-positive gastric carinomas. A novel in vivo yeast-based approach and in silico predictions were used. Variant E433Q was characterized for the first time and was shown to have no effect on MLH1 protein function. Functional analysis of amino acid rearrangement K618A, with previously reported contradictory results of its pathogenicity, suggests that the variant is a neutral polymorphism. Results of our study imply that there is either germline mutation or an epigenetic inactivation of another MMR gene, which causes MSI phenotype in the referred gastric cancer cases.