The EMT-activator ZEB1 is unrelated to platinum drug resistance in ovarian cancer but is predictive of survival.

The IGROVCDDP cisplatin-resistant ovarian cancer cell line is an unusual model, as it is also cross-resistant to paclitaxel. IGROVCDDP, therefore, models the resistance phenotype of serous ovarian cancer patients who have failed frontline platinum/taxane chemotherapy. IGROVCDDP has also undergone epithelial-mesenchymal transition (EMT). We aim to determine if alterations in EMT-related genes are related to or independent from the drug-resistance phenotypes. EMT gene and protein markers, invasion, motility and morphology were investigated in IGROVCDDP and its parent drug-sensitive cell line IGROV-1. ZEB1 was investigated by qPCR, Western blotting and siRNA knockdown. ZEB1 was also investigated in publicly available ovarian cancer gene-expression datasets. IGROVCDDP cells have decreased protein levels of epithelial marker E-cadherin (6.18-fold, p = 1.58e-04) and higher levels of mesenchymal markers vimentin (2.47-fold, p = 4.43e-03), N-cadherin (4.35-fold, p = 4.76e-03) and ZEB1 (3.43-fold, p = 0.04). IGROVCDDP have a spindle-like morphology consistent with EMT. Knockdown of ZEB1 in IGROVCDDP does not lead to cisplatin sensitivity but shows a reversal of EMT-gene signalling and an increase in cell circularity. High ZEB1 gene expression (HR = 1.31, n = 2051, p = 1.31e-05) is a marker of poor overall survival in high-grade serous ovarian-cancer patients. In contrast, ZEB1 is not predictive of overall survival in high-grade serous ovarian-cancer patients known to be treated with platinum chemotherapy. The increased expression of ZEB1 in IGROVCDDP appears to be independent of the drug-resistance phenotypes. ZEB1 has the potential to be used as biomarker of overall prognosis in ovarian-cancer patients but not of platinum/taxane chemoresistance.

The induction of a mesenchymal phenotype by platelet cloaking of cancer cells is a universal phenomenon.

Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions. The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model. Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR. Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.

The role of the MAD2-TLR4-MyD88 axis in paclitaxel resistance in ovarian cancer.

Despite the use of front-line anticancer drugs such as paclitaxel for ovarian cancer treatment, mortality rates have remained almost unchanged for the past three decades and the majority of patients will develop recurrent chemoresistant disease which remains largely untreatable. Overcoming chemoresistance or preventing its onset in the first instance remains one of the major challenges for ovarian cancer research. In this study, we demonstrate a key link between senescence and inflammation and how this complex network involving the biomarkers MAD2, TLR4 and MyD88 drives paclitaxel resistance in ovarian cancer. This was investigated using siRNA knockdown of MAD2, TLR4 and MyD88 in two ovarian cancer cell lines, A2780 and SKOV-3 cells and overexpression of MyD88 in A2780 cells. Interestingly, siRNA knockdown of MAD2 led to a significant increase in TLR4 gene expression, this was coupled with the development of a highly paclitaxel-resistant cell phenotype. Additionally, siRNA knockdown of MAD2 or TLR4 in the serous ovarian cell model OVCAR-3 resulted in a significant increase in TLR4 or MAD2 expression respectively. Microarray analysis of SKOV-3 cells following knockdown of TLR4 or MAD2 highlighted a number of significantly altered biological processes including EMT, complement, coagulation, proliferation and survival, ECM remodelling, olfactory receptor signalling, ErbB signalling, DNA packaging, Insulin-like growth factor signalling, ion transport and alteration of components of the cytoskeleton. Cross comparison of the microarray data sets identified 7 overlapping genes including MMP13, ACTBL2, AMTN, PLXDC2, LYZL1, CCBE1 and CKS2. These results demonstrate an important link between these biomarkers, which to our knowledge has never before been shown in ovarian cancer. In the future, we hope that triaging patients into alterative treatment groups based on the expression of these three biomarkers or therapeutic targeting of the mechanisms they are involved in will lead to improvements in patient outcome and prevent the development of chemoresistance.

Prostate cancer-derived holoclones: a novel and effective model for evaluating cancer stemness.

Prostate cancer accounts for approximately 13.5% of all newly diagnosed male cancer cases. Significant clinical burdens remain in terms of ineffective prognostication, with overtreatment of insignificant disease. Additionally, the pathobiology underlying disease heterogeneity remains poorly understood. As the role of cancer stem cells in the perpetuation of aggressive carcinoma is being substantiated by experimental evidence, it is crucially important to understand the molecular mechanisms, which regulate key features of cancer stem cells. We investigated two methods for in vitro cultivation of putative prostate cancer stem cells based on 'high-salt agar' and 'monoclonal cultivation'. Data demonstrated 'monoclonal cultivation' as the superior method. We demonstrated that 'holoclones' expressed canonical stem markers, retained the exclusive ability to generate poorly differentiated tumours in NOD/SCID mice and possessed a unique mRNA-miRNA gene signature. miRNA:Target interactions analysis visualised potentially critical regulatory networks, which are dysregulated in prostate cancer holoclones. The characterisation of this tumorigenic population lays the groundwork for this model to be used in the identification of proteomic or small non-coding RNA therapeutic targets for the eradication of this critical cellular population. This is significant, as it provides a potential route to limit development of aggressive disease and thus improve survival rates.

Circular RNAs are differentially expressed in prostate cancer and are potentially associated with resistance to enzalutamide.

Most forms of castration-resistant prostate cancer (CRPC) are dependent on the androgen receptor (AR) for survival. While, enzalutamide provides a substantial survival benefit, it is not curative and many patients develop resistance to therapy. Although not yet fully understood, resistance can develop through a number of mechanisms, such as AR copy number gain, the generation of splice variants such as AR-V7 and mutations within the ligand binding domain (LBD) of the AR. circular RNAs (circRNAs) are a novel type of non-coding RNA, which can regulate the function of miRNA, and may play a key role in the development of drug resistance. circRNAs are highly resistant to degradation, are detectable in plasma and, therefore may serve a role as clinical biomarkers. In this study, AR-V7 expression was assessed in an isogenic model of enzalutamide resistance. The model consisted of age matched control cells and two sub-line clones displaying varied resistance to enzalutamide. circRNA profiling was performed on the panel using a high throughout microarray assay. Bioinformatic analysis identified a number of differentially expressed circRNAs and predicted five miRNA binding sites for each circRNA. miRNAs were stratified based on known associations with prostate cancer, and targets were validated using qPCR. Overall, circRNAs were more often down regulated in resistant cell lines compared with control (588 vs. 278). Of particular interest was hsa_circ_0004870, which was down-regulated in enzalutamide resistant cells (p ≤ 0.05, vs. sensitive cells), decreased in cells that highly express AR (p ≤ 0.01, vs. AR negative), and decreased in malignant cells (p ≤ 0.01, vs. benign). The associated parental gene was identified as RBM39, a member of the U2AF65 family of proteins. Both genes were down-regulated in resistant cells (p < 0.05, vs. sensitive cells). This is one of the first studies to profile and demonstrate discrete circRNA expression patterns in an enzalutamide resistant cell line model of prostate cancer. Our data suggests that hsa_circ_0004870, through RBM39, may play a critical role in the development of enzalutamide resistance in CRPC.

CD10-/ALDH- cells are the sole cisplatin-resistant component of a novel ovarian cancer stem cell hierarchy.

It is long established that tumour-initiating cancer stem cells (CSCs) possess chemoresistant properties. However, little is known of the mechanisms involved, particularly with respect to the organisation of CSCs as stem-progenitor-differentiated cell hierarchies. Here we aimed to elucidate the relationship between CSC hierarchies and chemoresistance in an ovarian cancer model. Using a single cell-based approach to CSC discovery and validation, we report a novel, four-component CSC hierarchy based around the markers cluster of differentiation 10 (CD10) and aldehyde dehydrogenase (ALDH). In a change to our understanding of CSC biology, resistance to chemotherapy drug cisplatin was found to be the sole property of CD10-/ALDH- CSCs, while all four CSC types were sensitive to chemotherapy drug paclitaxel. Cisplatin treatment quickly altered the hierarchy, resulting in a three-component hierarchy dominated by the cisplatin-resistant CD10-/ALDH- CSC. This organisation was found to be hard-wired in a long-term cisplatin-adapted model, where again CD10-/ALDH- CSCs were the sole cisplatin-resistant component, and all CSC types remained paclitaxel-sensitive. Molecular analysis indicated that cisplatin resistance is associated with inherent- and adaptive-specific drug efflux and DNA-damage repair mechanisms. Clinically, low CD10 expression was consistent with a specific set of ovarian cancer patient samples. Collectively, these data advance our understanding of the relationship between CSC hierarchies and chemoresistance, which was shown to be CSC- and drug-type specific, and facilitated by specific and synergistic inherent and adaptive mechanisms. Furthermore, our data indicate that primary stage targeting of CD10-/ALDH- CSCs in specific ovarian cancer patients in future may facilitate targeting of recurrent disease, before it ever develops.

MyD88 is an essential component of retinoic acid-induced differentiation in human pluripotent embryonal carcinoma cells.

We have previously reported that myeloid differentiation primary response gene 88 (MyD88) is downregulated during all-trans retinoic acid (RA)-induced differentiation of pluripotent NTera2 human embryonal carcinoma cells (hECCs), whereas its maintained expression is associated with RA differentiation resistance in nullipotent 2102Ep hECCs. MyD88 is the main adapter for toll-like receptor (TLR) signalling, where it determines the secretion of chemokines and cytokines in response to pathogens. In this study, we report that loss of MyD88 is essential for RA-facilitated differentiation of hECCs. Functional analysis using a specific MyD88 peptide inhibitor (PepInh) demonstrated that high MyD88 expression in the self-renewal state inhibits the expression of a specific set of HOX genes. In NTera2 cells, MyD88 is downregulated during RA-induced differentiation, a mechanism that could be broadly replicated by MyD88 PepInh treatment of 2102Ep cells. Notably, MyD88 inhibition transitioned 2102Ep cells into a stable, self-renewing state that appears to be primed for differentiation upon addition of RA. At a molecular level, MyD88 inhibition combined with RA treatment upregulated HOX, RA signalling and TLR signalling genes. These events permit differentiation through a standard downregulation of Oct4-Sox2-Nanog mechanism. In line with its role in regulating secretion of specific proteins, conditioned media experiments demonstrated that differentiated (MyD88 low) NTera2 cell media was sufficient to differentiate NTera2 cells. Protein array analysis indicated that this was owing to secretion of factors known to regulate angiogenesis, neurogenesis and all three branches of TGF-ß Superfamily signalling. Collectively, these data offer new insights into RA controlled differentiation of pluripotent cells, with notable parallels to the ground state model of embryonic stem cell self-renewal. These data may provide insights to facilitate improved differentiation protocols for regenerative medicine and differentiation-therapies in cancer treatment.

Pluripotency markers are differentially induced by MEK inhibition in thyroid and melanoma BRAFV600E cell lines.

Oncogenic mutations in BRAF are common in melanoma and thyroid carcinoma and drive constitutive activation of the MAPK pathway. Molecularly targeted therapies of this pathway improves survival compared to chemotherapy; however, responses tend to be short-lived as resistance invariably occursCell line models of melanoma and thyroid carcinoma, +/- BRAF(V600E) activating mutation, were treated with the MEK inhibitor PD0325901. Treated and naive samples were assayed for expression of key members of the MAPK pathway. Global microRNA expression profiling of naive and resistant cells was performed via next generation sequencingand indicated pluripotency pathways in resistance. Parental cell lines were progressed to holoclones to confirm the miRNA stemness profileMembers of the MIR302/373/374/520 family of embryonic stem cell specific cell cycle regulating (ESCC) microRNAs were identified as differentially expressed between resistant BRAF(V600E) melanoma and thyroid cell lines. Upregulated expression of gene and protein stemness markers, upregulated expression of MAPK pathway genes and downregulation of the ESCC MIR302 cluster in BRAF(V600E) melanoma indicated an increased stem-like phenotype in resistant BRAF(V600E) melanoma. Conversely, downregulated expression of gene and protein stemness markers, downregulated expression of MAPK pathway genes, upregulation of the ESCC MIR520 cluster, reeexpression of cell surface receptors, and induced differentiation-associated morphology in resistant BRAF(V600E) indicate a differentiated phenotype associated with MEK inhibitor resistance in BRAF(V600E) thyroid cellsThe differential patterns of resistance observed between BRAF(V600E) melanoma and thyroid cell lines may reflect tissue type or de novo differentiation, but could have significant impact on the response of primary and metastatic cells to MEK inhibitor treatment. This study provides a basis for the investigation of the cellular differentiation/self-renewal access and its role in resistance to MEK inhibition.

Interleukin-15 is associated with disease severity in viral bronchiolitis.

Disease severity in viral bronchiolitis in infancy is difficult to predict and has been linked to host innate immunity. The study aimed to investigate the innate cytokine interleukin-15 (IL-15) as a marker of disease severity.A prospective single-centre observational study was conducted in a university-affiliated paediatric teaching hospital, comparing children (0-18 months) hospitalised for viral bronchiolitis, those admitted to the paediatric intensive care unit with severe disease and healthy age-matched controls. IL-15-related parameters were compared between groups. PCR and microRNA (miRNA) sequencing was undertaken on natural killer (NK) cells collected from study participants.Samples from 88 children with viral bronchiolitis and 43 controls enrolled between 2009 and 2012 were analysed. Peripheral blood mononuclear cell (PBMC) IL-15 mRNA expression was significantly higher in those with moderate severity bronchiolitis compared with controls and those with severe disease. Serum IL-15 levels correlated with disease severity. The relative frequency of NK cells in peripheral blood was significantly reduced in participants with bronchiolitis. The NK cell miRNA transcriptome in bronchiolitis was distinct. Targets of de-regulated miRNA were differentially expressed in bronchiolitis, including JAK3, STAT5A and NFKB1 on the IL-15 signalling pathway.IL-15 is associated with disease severity in children hospitalised with viral bronchiolitis.

Identifying novel hypoxia-associated markers of chemoresistance in ovarian cancer.

BACKGROUND: Ovarian cancer is associated with poor long-term survival due to late diagnosis and development of chemoresistance. Tumour hypoxia is associated with many features of tumour aggressiveness including increased cellular proliferation, inhibition of apoptosis, increased invasion and metastasis, and chemoresistance, mostly mediated through hypoxia-inducible factor (HIF)-1α. While HIF-1α has been associated with platinum resistance in a variety of cancers, including ovarian, relatively little is known about the importance of the duration of hypoxia. Similarly, the gene pathways activated in ovarian cancer which cause chemoresistance as a result of hypoxia are poorly understood. This study aimed to firstly investigate the effect of hypoxia duration on resistance to cisplatin in an ovarian cancer chemoresistance cell line model and to identify genes whose expression was associated with hypoxia-induced chemoresistance. METHODS: Cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) ovarian cancer cell lines were exposed to various combinations of hypoxia and/or chemotherapeutic drugs as part of a 'hypoxia matrix' designed to cover clinically relevant scenarios in terms of tumour hypoxia. Response to cisplatin was measured by the MTT assay. RNA was extracted from cells treated as part of the hypoxia matrix and interrogated on Affymetrix Human Gene ST 1.0 arrays. Differential gene expression analysis was performed for cells exposed to hypoxia and/or cisplatin. From this, four potential markers of chemoresistance were selected for evaluation in a cohort of ovarian tumour samples by RT-PCR. RESULTS: Hypoxia increased resistance to cisplatin in A2780 and A2780cis cells. A plethora of genes were differentially expressed in cells exposed to hypoxia and cisplatin which could be associated with chemoresistance. In ovarian tumour samples, we found trends for upregulation of ANGPTL4 in partial responders and down-regulation in non-responders compared with responders to chemotherapy; down-regulation of HER3 in partial and non-responders compared to responders; and down-regulation of HIF-1α in non-responders compared with responders. CONCLUSION: This study has further characterized the relationship between hypoxia and chemoresistance in an ovarian cancer model. We have also identified many potential biomarkers of hypoxia and platinum resistance and provided an initial validation of a subset of these markers in ovarian cancer tissues.

A novel serum microRNA panel to discriminate benign from malignant ovarian disease.

Ovarian cancer is the seventh most common cancer in women and the most frequent cause of gynaecological malignancy-related mortality in women. Currently, no standardized reliable screening test exists. MicroRNA profiling has allowed the identification of signatures associated with diagnosis, prognosis and response to treatment of human tumours. The aim of this study was to determine if a microRNA signature could distinguish between malignant and benign ovarian disease. A training set of 5 serous ovarian carcinomas and 5 benign serous cystadenomas were selected for the initial experiments. The validation set included 20 serous ovarian carcinomas and 20 benign serous cystadenomas. The serum/plasma focus microRNA Exiqon panel was used for the training set. For the validation set a pick and mix Exiqon panel, which focuses on microRNAs of interest was used. A panel of 4 microRNAs (let-7i-5p, miR-122, miR-152-5p and miR-25-3p) was significantly down regulated in cancer patients. These microRNAs target WNT signalling, AKT/mTOR and TLR-4/MyD88, which have previously been found to play a role in ovarian carcinogenesis and chemoresistance. let-7i-5p, miR-122, miR-152-5p and miR-25-3p could act as diagnostic biomarkers in ovarian cancer.

An integrated analysis of the SOX2 microRNA response program in human pluripotent and nullipotent stem cell lines.

BACKGROUND: SOX2 is a core component of the transcriptional network responsible for maintaining embryonal carcinoma cells (ECCs) in a pluripotent, undifferentiated state of self-renewal. As such, SOX2 is an oncogenic transcription factor and crucial cancer stem cell (CSC) biomarker in embryonal carcinoma and, as more recently found, in the stem-like cancer cell component of many other malignancies. SOX2 is furthermore a crucial factor in the maintenance of adult stem cell phenotypes and has additional roles in cell fate determination. The SOX2-linked microRNA (miRNA) transcriptome and regulome has not yet been fully defined in human pluripotent cells or CSCs. To improve our understanding of the SOX2-linked miRNA regulatory network as a contribution to the phenotype of these cell types, we used high-throughput differential miRNA and gene expression analysis combined with existing genome-wide SOX2 chromatin immunoprecipitation (ChIP) data to map the SOX2 miRNA transcriptome in two human embryonal carcinoma cell (hECC) lines. RESULTS: Whole-microRNAome and genome analysis of SOX2-silenced hECCs revealed many miRNAs regulated by SOX2, including several with highly characterised functions in both cancer and embryonic stem cell (ESC) biology. We subsequently performed genome-wide differential expression analysis and applied a Monte Carlo simulation algorithm and target prediction to identify a SOX2-linked miRNA regulome, which was strongly enriched with epithelial-to-mesenchymal transition (EMT) markers. Additionally, several deregulated miRNAs important to EMT processes had SOX2 binding sites in their promoter regions. CONCLUSION: In ESC-like CSCs, SOX2 regulates a large miRNA network that regulates and interlinks the expression of crucial genes involved in EMT.