The expression pattern of circulating miR-590-3p as a promising diagnostic biomarker for early detection of epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is the most common type of ovarian tumors. The biomarkers, which are being used for EOC screening, have low sensitivity and specificity leading to late diagnosis and high mortality rate. Thus, identification of effective biomarkers for early diagnosis of ovarian cancer has become a high priority in research. Here we aim to address this problem by studying the expression of a potential molecular marker, miR-590-3p, in EOC. We examined the mRNA steady-state levels of various acknowledged biomarkers including cancer antigen-125 (CA125) which is being used for EOC screening, c-reactive protein (CRP) which expression was correlated with EOC stage and paired homeobox 2 gene (PAX2) whose expression is correlated with the histological grade to characterize the histo-pathological features of the specimens at the molecular level. We examined the expression of miR-590-3p in EOC patients’ serum and EOC tissues, using real-time PCR. The levels of circulating miR-590-3p showed to be significantly elevated in 84.6% of the EOC patients’ serum, with 76.92% sensitivity and 85.7% specificity at its optimal cutoff value. Tissue miR-590-3p showed to be upregulated in EOC tissues compared to normal ovarian tissues and highly correlated with high-grade poorly differentiated EOC. The adoption of the epithelial-mesenchymal transition biomarkers was enriched in EOC with high miR-590-3p levels. The potential downstream target genes, SOX2, LEF1 and PAX2, were predicted using miRanda and Targetscan in silco tools and their expression profiles were in silco examined using Oncomine data-mining platform and StarBase Pan-cancer analysis. We examined the levels of SOX2, LEF1 and PAX2 at the RNA and the protein levels via semi-quantitative PCR and western blot, respectively. Among the investigated potential target genes, only PAX2 showed negative correlation with the levels of miR-590-3p in EOC tissues, suggesting that miR-590-3p could acquire its role in EOC carcinogenesis through regulation of cellular differentiation in EOC

Comprehensive transcriptomic analysis of cell lines as models of primary tumors across 22 tumor types.

Cancer cell lines are a cornerstone of cancer research but previous studies have shown that not all cell lines are equal in their ability to model primary tumors. Here we present a comprehensive pan-cancer analysis utilizing transcriptomic profiles from The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia to evaluate cell lines as models of primary tumors across 22 tumor types. We perform correlation analysis and gene set enrichment analysis to understand the differences between cell lines and primary tumors. Additionally, we classify cell lines into tumor subtypes in 9 tumor types. We present our pancreatic cancer results as a case study and find that the commonly used cell line MIA PaCa-2 is transcriptionally unrepresentative of primary pancreatic adenocarcinomas. Lastly, we propose a new cell line panel, the TCGA-110-CL, for pan-cancer studies. This study provides a resource to help researchers select more representative cell line models

Development and application of two novel monoclonal antibodies against overexpressed CD26 and integrin α3 in human pancreatic cancer.

Monoclonal antibody (mAb) technology is an excellent tool for the discovery of overexpressed cell surface tumour antigens and the development of targeting agents. Here, we report the development of two novel mAbs against CFPAC-1 human pancreatic cancer cells. Using ELISA, flow cytometry, immunoprecipitation, mass spectrometry, Western blot and immunohistochemistry, we found that the target antigens recognised by the two novel mAbs KU44.22B and KU44.13A, are integrin α3 and CD26 respectively, with high levels of expression in human pancreatic and other cancer cell lines and human pancreatic cancer tissue microarrays. Treatment with naked anti-CD26 mAb KU44.13A did not have any effect on the growth and migration of cancer cells nor did it induce receptor downregulation. In contrast, treatment with anti-integrin α3 mAb KU44.22B inhibited growth in vitro of Capan-2 cells, increased migration of BxPC-3 and CFPAC-1 cells and induced antibody internalisation. Both novel mAbs are capable of detecting their target antigens by immunohistochemistry but not by Western blot. These antibodies are excellent tools for studying the role of integrin α3 and CD26 in the complex biology of pancreatic cancer, their prognostic and predictive values and the therapeutic potential of their humanised and/or conjugated versions in patients whose tumours overexpress integrin α3 or CD26

Rainbow Kaposi's Sarcoma-Associated Herpesvirus Revealed Heterogenic Replication with Dynamic Gene Expression.

Molecular mechanisms of Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named "Rainbow-KSHV," that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication.IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented

The non-coding landscape of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease marked by frequent recurrence and metastasis and stagnant survival rates. To enhance molecular knowledge of HNSCC and define a non-coding RNA (ncRNA) landscape of the disease, we profiled the transcriptome-wide dysregulation of long non-coding RNA (lncRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA) using RNA-sequencing data from 422 HNSCC patients in The Cancer Genome Atlas (TCGA). 307 non-coding transcripts differentially expressed in HNSCC were significantly correlated with patient survival, and associated with mutations in TP53, CDKN2A, CASP8, PRDM9, and FBXW7 and copy number variations in chromosomes 3, 5, 7, and 18. We also observed widespread ncRNA correlation to concurrent TP53 and chromosome 3p loss, a compelling predictor of poor prognosis in HNSCCs. Three selected ncRNAs were additionally associated with tumor stage, HPV status, and other clinical characteristics, and modulation of their expression in vitro reveals differential regulation of genes involved in epithelial-mesenchymal transition and apoptotic response. This comprehensive characterization of the HNSCC non-coding transcriptome introduces new layers of understanding for the disease, and nominates a novel panel of transcripts with potential utility as prognostic markers or therapeutic targets

The clinical and prognostic use of circulating tumour cells in breast cancer

Adjuvant therapies such as endocrine or cytotoxic chemotherapy have been demonstrated to improve overall survival in early breast cancer patients. A blood test to monitor patients at risk of relapse is needed to identify those patients who would benefit from these treatments and those for whom it is not necessary. This is in favour of detecting disseminated tumour cells (DTCs) from painful bone marrow aspirates, currently the gold‐standard method for detecting minimal residual disease (MRD). The use of circulating tumour cells (CTCs) enriched from the blood was investigated for this purpose along with their characterisation in the metastatic setting to enable individualised therapy. Sixty‐four primary breast cancer patients were followed up for up to 12 years post surgery for any MRD present. This analysis looked at measurements of DTCs in the bone marrow, CTCs in the blood and circulating‐free DNA (cfDNA) in the plasma over the follow up period. Patients who had involved lymph nodes at surgery, were significantly more likely to have CTCs present than low risk patients with no nodes positive, (70% compared to 39% respectively, p = 0.042). Our analysis also looked at the relationship of cfDNA to DTCs and CTCs. An inverse relationship of cell death in the blood (manifesting as blood cfDNA) to bone marrow DTCs by qRT‐PCR was apparent. This may be due to tumour dormancy mechanisms ‐ cycles of tumour cell proliferation and cell death occurring in the bone marrow, evidence not shown before in patient samples. Combined use of these markers could therefore be used as a monitoring system for impending metastatic disease and a rationale for further treatment. We also participated in a multi–centre study to assess the effects of lapatinib; a targeted therapy against two members of the human epidermal growth factor receptor family (EGFR and HER2). This was in advanced breast cancer patients and used CTCs as a surrogate marker. Our study selected patients on the basis of EGFR positivity in CTCs that were present in the blood. Four out of 12 patients (33%) demonstrated an initial decrease in the number of EGFR positive CTCs in response to Lapatinib, however this was limited and all patients were taken off study with progressive disease. We also explored a novel method in development to detect viable CTCs. This used an in situ hybridisation method to amplify signals from mRNA transcripts of tumour markers in CTCs. The use of CTCs is a very useful and promising tool for studying both the biology of breast cancer, and also as a non‐invasive analytical tool in the clinical setting to gain predictive and prognostic information

A human lung tumor microenvironment interactome identifies clinically relevant cell-type cross-talk.

BackgroundTumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway.ResultTo develop a deeper understanding of the interactions between cells within human lung tumors, we perform RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We map the cell-specific differential expression of prognostically associated secreted factors and cell surface genes, and computationally reconstruct cross-talk between these cell types to generate a novel resource called the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identify and validate a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also find a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior.ConclusionThese results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance

Microchips and their significance in isolation of circulating tumor cells and monitoring of cancers

In micro-fluid systems, fluids are injected into extremely narrow polymer channels in small amounts such as micro-, nano-, or pico-liter scales. These channels themselves are embedded on tiny chips. Various specialized structures in the chips including pumps, valves, and channels allow the chips to accept different types of fluids to be entered the channel and along with flowing through the channels, exert their effects in the framework of different reactions. The chips are generally crystal, silicon, or elastomer in texture. These highly organized structures are equipped with discharging channels through which products as well as wastes of the reactions are secreted out. A particular advantage regarding the use of fluids in micro-scales over macro-scales lies in the fact that these fluids are much better processed in the chips when they applied as micro-scales. When the laboratory is miniaturized as a microchip and solutions are injected on a micro-scale, this combination makes a specialized construction referred to as "lab-on-chip". Taken together, micro-fluids are among the novel technologies which further than declining the costs; enhancing the test repeatability, sensitivity, accuracy, and speed; are emerged as widespread technology in laboratory diagnosis. They can be utilized for monitoring a wide spectrum of biological disorders including different types of cancers. When these microchips are used for cancer monitoring, circulatory tumor cells play a fundamental role

The interplay between obesity and cancer: a fly view

Accumulating epidemiological evidence indicates a strong clinical association between obesity and an increased risk of cancer. The global pandemic of obesity indicates a public health trend towards a substantial increase in cancer incidence and mortality. However, the mechanisms that link obesity to cancer remain incompletely understood. The fruit fly Drosophila melanogaster has been increasingly used to model an expanding spectrum of human diseases. Fly models provide a genetically simpler system that is ideal for use as a first step towards dissecting disease interactions. Recently, the combining of fly models of diet-induced obesity with models of cancer has provided a novel model system in which to study the biological mechanisms that underlie the connections between obesity and cancer. In this Review, I summarize recent advances, made using Drosophila, in our understanding of the interplay between diet, obesity, insulin resistance and cancer. I also discuss how the biological mechanisms and therapeutic targets that have been identified in fly studies could be utilized to develop preventative interventions and treatment strategies for obesityassociated cancers

GBM heterogeneity as a function of variable epidermal growth factor receptor variant III activity.

Abnormal activation of the epidermal growth factor receptor (EGFR) due to a deletion of exons 2-7 of EGFR (EGFRvIII) is a common alteration in glioblastoma (GBM). While this alteration can drive gliomagenesis, tumors harboring EGFRvIII are heterogeneous. To investigate the role for EGFRvIII activation in tumor phenotype we used a neural progenitor cell-based murine model of GBM driven by EGFR signaling and generated tumor progenitor cells with high and low EGFRvIII activation, pEGFRHi and pEGFRLo. In vivo, ex vivo, and in vitro studies suggested a direct association between EGFRvIII activity and increased tumor cell proliferation, decreased tumor cell adhesion to the extracellular matrix, and altered progenitor cell phenotype. Time-lapse confocal imaging of tumor cells in brain slice cultures demonstrated blood vessel co-option by tumor cells and highlighted differences in invasive pattern. Inhibition of EGFR signaling in pEGFRHi promoted cell differentiation and increased cell-matrix adhesion. Conversely, increased EGFRvIII activation in pEGFRLo reduced cell-matrix adhesion. Our study using a murine model for GBM driven by a single genetic driver, suggests differences in EGFR activation contribute to tumor heterogeneity and aggressiveness