Comprehending the proteomic landscape of ovarian cancer : A road to the discovery of disease biomarkers

Despite recent technological advancements allowing the characterization of cancers at a molecular level along with biomarkers for cancer diagnosis, the management of ovarian cancers (OC) remains challenging. Proteins assume functions encoded by the genome and the complete set of proteins, termed the proteome, reflects the health state. Comprehending the circulatory proteomic profiles for OC subtypes, therefore, has the potential to reveal biomarkers with clinical utility concerning early diagnosis or to predict response to specific therapies. Furthermore, characterization of the proteomic landscape of tumor-derived tissue, cell lines, and PDX models has led to the molecular stratification of patient groups, with implications for personalized therapy and management of drug resistance. Here, we review single and multiple marker panels that have been identified through proteomic investigations of patient sera, effusions, and other biospecimens. We discuss their clinical utility and implementation into clinical practice

Multipronged quantitative proteomic analyses indicate modulation of various signal transduction pathways in human meningiomas

Meningiomas (MGs) are frequent tumors of the CNS originating from the meningeal layers of the spinal cord and the brain. In this study, comparative tissue proteomic analysis of low and high grades of MGs was performed by using iTRAQ-based quantitative proteomics in combination with ESI-quadrupole-TOF and Q-Exactive MS, and results were validated by employing ELISA. Combining the results obtained from two MS platforms, we were able to identify overall 4308 proteins (1% false discover rate), among which 2367 exhibited differential expression (more than and equal to 2 peptide and ≥1.5-fold in at least one grade) in MGs. Several differentially expressed proteins were found to be associated with diverse signaling pathways, including integrin, Wnt, Ras, epidermal growth factor receptor, and FGR signaling. Proteins, such as vinculin or histones, which act as the signaling activators to initiate multiple signaling pathways, were found to be upregulated in MGs. Quite a few candidates, such as protein S-100A6, aldehyde dehydrogenase mitochondrial, AHNAK, cytoskeleton-associated protein 4, and caveolin, showed sequential increase in low- and high-grade MGs, whereas differential expressions of collagen alpha-1 (VI), protein S100-A9, 14 kDa phosphohistidine phosphatase, or transgelin-2 were found to be grade specific. Our findings provide new insights regarding the association of various signal transduction pathways in MG pathogenesis and may introduce new opportunities for the early detection and prognosis of MGs

A single nucleotide polymorphism associated with hepatitis C virus infections located in the distal region of the IL28B promoter influences NF-κB-mediated gene transcription.

Persistence of hepatitis C virus (HCV) infection is observed only in a subset of infected individuals and among them only some respond to treatment. Genome-wide association studies (GWAS) carried out around the world identified single nucleotide polymorphisms (SNPs) in the IL28B locus that are strongly associated with both HCV clearance and treatment response. The functional significance of these associations however, is not clear. In this report we show that an SNP rs28416813 in the distal promoter region of IL28B that is in close proximity to a non-consensus NF-κB-binding site affects downstream reporter gene expression. The effect is likely due to differential binding of NF-κB at the non-consensus site. The non-protective allele showed a reduction in luciferase reporter gene expression compared to the protective allele in HEK293T cells under different experimental conditions including treatment with tumor necrosis factor alpha (TNF-α) and 5' triphosphorylated dsRNA. Furthermore, the HCV RNA polymerase was able to induce transcription from the IL28B promoter in a RIG-I-dependent manner. This induction was influenced by the alleles present at rs28416813. We also demonstrate strong linkage disequilibrium between rs28416813 and another important SNP rs12979860 in two ethnic populations. These results suggest possible mechanisms by which SNPs at the IL28B locus influence spontaneous clearance and treatment response in chronic HCV infections

Comprehensive proteomic analysis reveals distinct functional modules associated with skull base and supratentorial meningiomas and perturbations in collagen pathway components

Meningiomas are brain tumors that originate from the meninges and has been primarily classified into three grades by the current WHO guidelines. Although widely prevalent and can be managed by surgery there are instances when the tumors are located in difficult regions. This results in considerable challenges for complete surgical resection and further clinical management. While the genetic signature of the skull base tumors is now known to be different from the non-skull base tumors, there is a lack of information at the functional aspects of these tumors at the proteomic level. Thus, the current study thereby aims to obtain mechanistic insights between the two radiologically distinct groups of meningiomas, namely the skull base & supratentorial (non-skull base-NSB) regions. We have employed a comprehensive mass spectrometry-based label-free quantitative proteomic analysis in Skull base and supratentorial meningiomas. Further, we have used an Artificial Neural Networking employing a sparse Multilayer perceptron (MLP) architecture to predict protein concordance. A patient-derived spectral library has been employed for a novel peptide-level validation of proteins that are specific to the radiological regions using the SRM assay based targeted proteomics approach. The comprehensive proteomics enabled the identification of nearly 4000 proteins with high confidence (1%FDR ≥ 2 unique peptides) among which 170 proteins were differentially abundant in Skull base vs Supratentorial tumors (p-value ≤0.05). In silico analysis enabled mapping of the major alterations and hinted towards an overall perturbation of extracellular matrix and collagen biosynthesis components in the non-skull base meningiomas and a prominent perturbation of molecular trafficking in the skull base meningiomas. Therefore, this study has yielded novel insights into the functional association of the proteins that are differentially abundant in the two radiological subgroups. Significance: In the current study, we have performed label-free proteomic analysis on fresh frozen tissue of 14 Supratentorial (NSB) and 7 Skull base meningiomas to assess perturbations in the global proteome, we have further employed an in-depth in silico analysis to map the pathways that have enabled functional mapping of the differentially abundant proteins in the Skull base and Supratentorial tumors. The findings from the above were also subjected to a machine learning-based neural networking to find out the proteins that have the most concordance of occurrence to determine the most influential proteins of the network. We further validated the differential abundance of identified protein markers in a larger patient cohort of Skull base and Supratentorial employing targeted proteomics approach to validate key protein candidates emerging from ours and other recent studies. The previous studies that have explored the skull base and convexity meningiomas have been able to reveal alterations in the genetic mutations in these tumor types. However, there are not many studies that have explored the functional aspects of these tumors, especially at the proteome level. We have attempted for the first time to map the functional modules associated with altered proteins in these tumors and have been able to identify that there is a possibility that the Skull base meningiomas to be considerably different from the Non-skull base (NSB) tumors in terms of the perturbed pathways. Our study employed global as well as targeted proteomics to examine the proteomic alterations in these two tumor groups. The study indicates that proteins that were more abundant in Skull base tumors were part of molecular transport components, non-skull base proteins majorly mapped to the components of extracellular matrix remodeling pathways. In conclusion, this study substantiates the distinction in the proteomic signatures in the skull base and supratentorial meningiomas paving way for further investigation of the identified markers for determining if some of these proteins can be used for therapeutic interventions for cases that pose considerable challenges for complete resection

The non-protective C allele at rs28416813-rev reduces transcription of reporter gene likely by decreasing NF-κB binding at the non-consensus site.

<p>A) A schematic of the ΔNF construct made with the p1.4IL28B construct with G allele at rs28416813-rev. TSS-transcription start site. The grey rectangle represents the non-consensus NF-κB-binding site between +115 to +124. The vertical line next to the grey rectangle denotes the SNP rs28416813-rev. The dashed line denotes the deletion made between +115 and +163 to generate ΔNF construct. The sequences of the other constructs used are shown in the right with the non-cosensus NF-κB site underlined and the SNP rs28416813-rev in italics. B) A representative plot (of at least two independent experiments, error bars show SD of triplicates) of luciferase activities of p1.4IL28B constructs with G or C alleles at rs28416813-rev and with different mutations introduced in to the non-consensus NF-κB site at +115 position to the plasmid with G allele background.</p

rs28416813 is in LD with rs12979860.

<p>A) EtBr-stained gel of the PCR products that were amplified from the <i>IL28A</i> and <i>IL28B</i> promoters that included the SNP rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075495#pone.0075495-Smith1" target="_blank">[35]</a>. The ∼2 kb fragment specific to <i>IL28B</i> was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075495#pone.0075495-Shin1" target="_blank">[20]</a>.</p

The distal fragment of the <i>IL28B</i> promoter is responsive to NF-κB.

<p>A) A schematic of the 1.4 kb fragment of the <i>IL28B</i> promoter fragment cloned in to pGL3basic vector. The rectangles show the different transcription factor binding sites identified by Osterlund et al. (2007) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075495#pone.0075495-Osterlund1" target="_blank">[21]</a>. The vertical lines next to the non-consensus NF-κB site indicate the position of two SNPs rs28416813 and rs71356849 respectively. ISRE, Interferon-stimulated response element, PRDI, positive-regulatory domain I. The TSS is shown by the arrow before the non-consensus NF-κB-binding site (at position +115 with respect to TSS). B) Luciferase assays carried out with HEK293T cells in 6-well plates. The transfection of the p1.4IL28BC-T construct was done either in presence or absence of p50+p65 and activity compared with the empty pGL3basic vector. The error bars show SD from triplicates.</p

TNF-α and 5′ppp-dsRNA stimulate transcription from <i>IL28B</i> promoter.

<p>A) p1.4IL28B constructs (10 ng/well) with either G or C alleles at rs28416813-rev along with pRLTK were transfected in to 96-well plates and after 24 hr recombinant human TNF-α was added in to the media at indicated concentrations and plates were incubated for a further 8 hr before subjecting to luciferase assays. The experiment was done at least twice with similar results and the error bars show SD from triplicates. B) and C) p1.4IL28B constructs (10 ng/well) with either G or C alleles at rs28416813-rev along with pRLTK and either pUNO (B) or pUNO-RIG-I (C) were transfected in to 96-well plates and after 24 hr a 19-mer 5′ppp-dsRNA was transfected at 250 nM concentration. Luciferase assays were done after 12 hr. The error bars show SD of 6 replicates from two experiments.</p

HCV RdRp can stimulate transcription from <i>IL28B</i> promoter.

<p>The gene encoding genotype 2a HCV RdRp (NS5B) was co-transfected at 50 ng/well along with the respective p1.4IL28B constructs at 10 ng/well and either RIG-I(WT) (A) RIG-I(K861E) (B) or MDA5 (C) at 0.5 ng/well <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075495#pone.0075495-RanjithKumar1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075495#pone.0075495-SubbaReddy1" target="_blank">[31]</a> along with pRLTK and luciferase assays were carried out after 36 hr. The experiment was repeated independently at least two times with similar results and the error bars show SD of triplicates from one experiment.</p

The C allele at rs28416813-rev in <i>IL28B</i> promoter decreases luciferase expression.

<p>A) Luciferase activity with p1.4IL28B constructs shows that the C allele at rs28416813-rev when present along with the T allele at rs71356849-rev decreases reporter gene expression. The ratio of firefly to <i>Renilla</i> luciferase values for the other three constructs was normalized to that of p1.4IL28BG-T construct as % activity and plotted. The data is representative of at least two independent experiments with the error bars showing SD of triplicates. B) A representative chromatogram from a small cohort of HCV infected patients (n = 25) who were sequenced at the two SNP positions (shown in bold) and only rs28416813-rev was dimorphic (C/G) with a minor allele frequency (MAF) of 0.3 whereas none of the samples showed any variation at rs71356849-rev (T). C) The C allele at rs28416813-rev consistently shows a reduction in luciferase activity by 20–30%. Results show the mean (76.5) of four independent experiments where samples were processed in triplicate and were controlled for transfection efficiency and were normalized to the G allele. Error bars show SE.</p