Exploiting the Stemness and Chemoresistance Transcriptome of Ewing Sarcoma to Identify Candidate Therapeutic Targets and Drug-Repurposing Candidates.

Outcomes for most patients with Ewing sarcoma (ES) have remained unchanged for the last 30 years, emphasising the need for more effective and tolerable treatments. We have hypothesised that using small-molecule inhibitors to kill the self-renewing chemotherapy-resistant cells (Ewing sarcoma cancer stem-like cells; ES-CSCs) responsible for progression and relapse could improve outcomes and minimise treatment-induced morbidities. For the first time, we demonstrate that ABCG1, a potential oncogene in some cancers, is highly expressed in ES-CSCs independently of CD133. Using functional models, transcriptomics and a bespoke in silico drug-repurposing pipeline, we have prioritised a group of tractable small-molecule inhibitors for further preclinical studies. Consistent with the cellular origin of ES, 21 candidate molecular targets of pluripotency, stemness and chemoresistance were identified. Small-molecule inhibitors to 13 of the 21 molecular targets (62%) were identified. POU5F1/OCT4 was the most promising new therapeutic target in Ewing sarcoma, interacting with 10 of the 21 prioritised molecular targets and meriting further study. The majority of small-molecule inhibitors (72%) target one of two drug efflux proteins, p-glycoprotein (n = 168) or MRP1 (n = 13). In summary, we have identified a novel cell surface marker of ES-CSCs and cancer/non-cancer drugs to targets expressed by these cells that are worthy of further preclinical evaluation. If effective in preclinical models, these drugs and drug combinations might be repurposed for clinical evaluation in patients with ES.

The angiotensin-converting enzyme gene insertion/deletion polymorphism interacts with fear of falling in relation to stepping speed in community-dwelling older adults.

BACKGROUND: Despite the association of genetic factors with falls, balance, and lower extremity functioning, interaction of the angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism with fear of falling (FOF) in relation to stepping performance has, to the best of our knowledge, not been investigated in older adults. OBJECTIVE: The purpose of this study was to examine the interaction effects of the ACE I/D polymorphism with FOF in relation to stepping performance in older adults. METHODS: Eighty-eight community-dwelling adults 60 years or older participated in a cross-sectional observational study. Participants completed tests of rapid and distance stepping, and self-reported FOF (yes/no). Participants provided saliva for ACE genotyping. General linear models evaluated ACE genotype × FOF interaction effects in relation to stepping performance. The α level was set at 0.05. RESULTS: The ACE I/D polymorphism exhibited significant interaction effects (p for interactions 0.002 ≤ p ≤ .04) with FOF in relation to stepping speed. Relationships between FOF and stepping speed varied among ACE genotypes. The insertion/insertion (II) genotype was significantly associated (p = .01) with slow stepping in individuals with, but not without FOF (p > .05). CONCLUSION: Variation in relationships between FOF and stepping speed among ACE genotypes suggests a role for the ACE I/D polymorphism in modifying relationships between FOF and stepping speed in older adults. The association of the ACE II genotype with slow stepping performance in individuals with, but not without FOF, suggests that older adults with the ACE II genotype and FOF may be at increased risk for poor stepping performance and associated functional declines.

Lectin Histochemistry: Historical Perspectives, State of the Art, and Future Directions.

Lectins, discovered more than 100 years ago and defined by their ability to selectively recognize specific carbohydrate structures, are ubiquitous in living organisms. Their precise functions are as yet under-explored and incompletely understood but they are clearly involved, through recognition of their binding partners, in a myriad of biological mechanisms involved in cell identity, adhesion, signaling, and growth regulation in health and disease. Understanding the complex "sugar code" represented by the "glycome" is a major challenge and at the forefront of current biological research. Lectins have been widely employed in histochemical studies to map glycosylation in cells and tissues. Here, a brief history of the discovery of lectins and early developments in their use is presented along with a selection of some of the most interesting and significant discoveries to emerge from the use of lectin histochemistry. Further, an evaluation of the next generation of lectin-based technologies is presented, including the potential for designing recombinant lectins with more precisely defined binding characteristics, linking lectin-based studies with other technologies to answer fundamental questions in glycobiology and approaches to exploring the interactions of lectins with their binding partners in more detail.

Utilising extracellular vesicles for early cancer diagnostics: benefits, challenges and recommendations for the future.

To increase cancer patient survival and wellbeing, diagnostic assays need to be able to detect cases earlier, be applied more frequently, and preferably before symptoms develop. The expansion of blood biopsy technologies such as detection of circulating tumour cells and cell-free DNA has shown clinical promise for this. Extracellular vesicles released into the blood from tumour cells may offer a snapshot of the whole of the tumour. They represent a stable and multifaceted complex of a number of different types of molecules including DNA, RNA and protein. These represent biomarker targets that can be collected and analysed from blood samples, offering great potential for early diagnosis. In this review we discuss the benefits and challenges of the use of extracellular vesicles in this context and provide recommendations on where this developing field should focus their efforts to bring future success.

RNA sequencing and functional studies of patient-derived cells reveal that neurexin-1 and regulators of this pathway are associated with poor outcomes in Ewing sarcoma.

PURPOSE: The development of biomarkers and molecularly targeted therapies for patients with Ewing sarcoma (ES) in order to minimise morbidity and improve outcome is urgently needed. Here, we set out to isolate and characterise patient-derived ES primary cell cultures and daughter cancer stem-like cells (CSCs) to identify biomarkers of high-risk disease and candidate therapeutic targets. METHODS: Thirty-two patient-derived primary cultures were established from treatment-naïve tumours and primary ES-CSCs isolated from these cultures using functional methods. By RNA-sequencing we analysed the transcriptome of ES patient-derived cells (n = 24) and ES-CSCs (n = 11) to identify the most abundant and differentially expressed genes (DEGs). Expression of the top DEG(s) in ES-CSCs compared to ES cells was validated at both RNA and protein levels. The functional and prognostic potential of the most significant gene (neurexin-1) was investigated using knock-down studies and immunohistochemistry of two independent tumour cohorts. RESULTS: ES-CSCs were isolated from all primary cell cultures, consistent with the premise that ES is a CSC driven cancer. Transcriptional profiling confirmed that these cells were of mesenchymal origin, revealed novel cell surface targets for therapy that regulate cell-extracellular matrix interactions and identified candidate drivers of progression and relapse. High expression of neurexin-1 and low levels of regulators of its activity, APBA1 and NLGN4X, were associated with poor event-free and overall survival rates. Knock-down of neurexin-1 decreased viable cell numbers and spheroid formation. CONCLUSIONS: Genes that regulate extracellular interactions, including neurexin-1, are candidate therapeutic targets in ES. High levels of neurexin-1 at diagnosis are associated with poor outcome and identify patients with localised disease that will relapse. These patients could benefit from more intensive or novel treatment modalities. The prognostic significance of neurexin-1 should be validated independently.

Multimodal analysis of cell-free DNA whole-genome sequencing for pediatric cancers with low mutational burden.

Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers.

Incorporating cytologic adequacy assessment into precision oncology workflow using telepathology: An institutional experience.

BACKGROUND: Tumor sample quality and quantity determine the success of somatic mutation analysis. Thus, a rapid on-site evaluation (ROSE) tumor cytology adequacy assessment was incorporated into the workflow of precision oncology at Weill Cornell Medicine in New York City. Optimal samples were obtained from 68 patients with metastatic cancer. METHODS: Cytopathologists performed ROSE on fine-needle aspirate samples via telepathology, and subsequently core-needle biopsies were obtained. In a retrospective manner, the concordance between adequacy assessment and the success rate of the procedure was evaluated to obtain sufficient tumor tissue for next-generation sequencing (NGS). RESULTS: Out of the 68 procedures, 43 were documented as adequate and 25 were documented as inadequate. The diagnostic yield of adequate procedures was 100%. Adequacy evaluation predicted the success rate of molecular profiling in 40 of 43 procedures (93%; 95% CI, 80.9-98.5 procedures). The success rate of molecular testing was significantly higher in the adequate group: 93% compared with 32% in the inadequate group (P < .0005). Seven procedures that failed to provide quality material for mutational analysis and pathological diagnosis were evaluated as inadequate. Cell block provided sufficient DNA for NGS in 6 cases. In 2 cases, a core biopsy could not be performed; hence, the fine-needle aspirate material confirmed the diagnosis and was used for NGS testing. CONCLUSION: These results support the incorporation of ROSE into the workflow of precision oncology to obtain high-quality tissue samples from metastatic lesions. In addition, NGS testing of concurrent cytology specimens with adequate cellularity can be a surrogate for NGS testing of biopsy specimens.

Influence of the breast prosthesis volume in dose distribution in radiotherapy planning

Introduction: The challenge of modern radiotherapy (RT) in breast cancer is to maintain its satisfactory oncological results, adapting to oncoplastic surgery and avoiding possible cosmetic damage. Considering that the breast prosthesis is not a target volume in RT planning, this study sought to analyze the effect of this volume on the coverage of the clinical target volume (CTV) of the breast. Methods: We performed a retrospective analysis of plans in 48 patients who submitted to RT in the first half of 2014. Two volumes were measured, such as breast CTV (breast tissue with the prosthesis) and real CTV (breast tissue excluding the prosthesis). The D95% values (dose that covers 95% of the volume) for each of them were verified and related to the volume of each one as well as the volume of breast prosthesis. Results: The analysis of the CTVs showed a significant difference between the mean volumes for the real CTV and breast CTV. While performing the CTV coverage, including the prosthesis, there is a perception that the dose covered 95% of the volume. Nevertheless, the analysis of the same plan after prosthesis volume exclusion revealed a difficulty in covering 95% of the breast tissue volume, indicating the interference of the prosthesis in therapy planning. Considering the dosimetric aspects, there were patients with real CTV values below the ideal dose of 47.5 Gy, after exclusion of implant volume. Conclusions: Our data reflected the volume of the prosthesis as an important variable that should be considered when planning adjuvant RT.

A practical toolkit to study aspects of the metastatic cascade in vitro.

While metastasis - the spread of cancer from the primary location to distant sites in the body - remains the principle cause of cancer death, it is incompletely understood. It is a complex process, requiring the metastatically successful cancer cell to negotiate a formidable series of interconnected steps, which are described in this paper. For each step, we review the range of in vitro assays that may be used to study them. We also provide a range of detailed, step-by-step protocols that can be undertaken in most modestly-equipped laboratories, including methods for converting qualitative observations into quantitative data for analysis. Assays include: (1) a gelatin degradation assay to study the ability of endothelial cells to degrade extracellular matrix during tumour angiogenesis; (2) the morphological characterisation of cells undergoing epithelial-mesenchymal transition (EMT) as they acquire motility; (3) a 'scratch' or 'wound-healing' assay to study cancer cell migration; (4) a transwell assay to study cancer cell invasion through extracellular matrix; and (5) a static adhesion assay to examine cancer cell interactions with, and adhesion to, endothelial monolayers. This toolkit of protocols will enable researchers who are interested in metastasis to begin to focus on defined aspects of the process. It is only by further understanding this complex, fascinating and clinically relevant series of events that we may ultimately devise ways of better treating, or even preventing, cancer metastasis. The assays may also be of more broad interest to researchers interested in studying aspects of cellular behaviour in relation to other developmental and disease processes.

Contemporary public image of the nursing profession in Saudi Arabia.

BACKGROUND: In the Kingdom of Saudi Arabia, the nursing profession faces significant challenges including; failure to recruit high school students into nursing education, poor nursing identity, and lack of awareness about the nursing profession. The level of community awareness and public image of the nursing profession are critical to recruit and retain students into nursing education, and to improve nurses' sense of identity. AIM: To explore the level of community awareness and public image of the nursing profession in Saudi Arabia. METHODS: We conducted a cross-sectional study with a convenient sample of 502 adults including106 males and 396 females, their mean age was 22.93 ± 6.76 years. Data collected included; socio-cultural data, gender preference in getting nursing care, awareness, and perceived socio-cultural barriers to pursue a nursing career. Data were analyzed using SPSS version 21.0. RESULTS: Only 32.5% preferred to get nursing care by Saudis. The nursing profession was not viewed as a respected job as 71.5% of participants would be ashamed of having a nurse in their families. The study revealed a low median knowledge score (50.0, IQR: 50.0-66.7)). The study highlighted a number of socio-cultural barriers to pursue a nursing career including; the gender-mixed working environment (35.9%), delayed marriage of female nurses (20.3%), and the negative effect of nursing profession on social life (64.5%). CONCLUSIONS: Half of the sample had a knowledge score below 50.0 out of 100. This level of poor awareness, in addition to socio-cultural perceived barriers are the main factors contributing to the negative public image of the nursing profession in Saudi Arabia. Understanding these factors could contribute to implementing focused intervention to improve the negative stereotype of the nursing profession among Saudis.

Angiotensin-Converting Enzyme Insertion/Deletion Polymorphism, Lower Extremity Strength, and Physical Performance in Older Adults.

BACKGROUND: Evidence for associations between the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene and physical performance is conflicting. Furthermore, investigations of relationships between lower extremity strength and physical performance have usually not considered the role of the ACE genotype, and it is unclear whether there are variations in relationships between lower extremity strength and physical performance among ACE genotypes in older adults. OBJECTIVE: The objectives of this study were to investigate associations between the ACE I/D polymorphism and physical performance and to determine whether relationships between lower extremity strength and physical performance vary among ACE genotypes in older adults. DESIGN: This was a cross-sectional observational study. METHODS: Community-dwelling adults (N = 88) who were at least 60 years old completed physical performance and lower extremity strength tests. After DNA was extracted from saliva, ACE I/D polymorphism genotyping was done. The Spearman rank order correlation coefficient was used to examine associations between lower extremity strength and physical performance within ACE genotype subgroups. Analysis of covariance and linear regression were used to examine ACE genotype and ACE genotype × lower extremity strength interaction effects in relation to physical performance. RESULTS: Genotype-specific correlation coefficients exhibited substantial variation among ACE genotype subgroups; however, differences did not attain statistical significance. Statistically significant genotype × lower extremity strength interaction effects in relation to physical performance were detected. LIMITATIONS: The cross-sectional design precludes inferring causal relationships between strength and performance. The small sample size contributed to limited power to detect additional interaction effects and to detect statistically significant differences between correlation coefficients among ACE genotype subgroups. CONCLUSIONS: The ACE I/D polymorphism is, interactively with lower extremity strength, associated with physical performance. Genotype-specific correlation coefficients and ACE genotype × lower extremity strength interaction effects on physical performance are consistent with variations in relationships between lower extremity strength and performance among ACE genotype subgroups.

ABCG1 and Pgp identify drug resistant, self-renewing osteosarcoma cells.

Patients with osteosarcoma (OST) frequently relapse with drug resistant disease, consistent with the hypothesis that tumours contain a cancer stem-like cell (CSCs) population that survives chemotherapy to re-populate the tumour at local or metastatic sites. We describe a dual functional approach to isolate OST-CSCs and identify the ABC transporter proteins driving this population to reveal potential targets for the development of new treatments. OST-CSCs were isolated by selection in doxorubicin (OST-EC50 cells) and based on the ability to produce progeny from a single cell (HOS-EC50.SR cells). Pgp expression was increased in OST-EC50 cells, inducing resistance to doxorubicin, etoposide, vincristine and actinomycin D (p < 0.05). Increased expression of ABCG1 and Pgp protein in the HOS-EC50.SR cells induced resistance to etoposide and doxorubicin (p < 0.01), which was directly correlated with ABCG1 expression (r > 0.88, p < 0.001). Pgp expression is increased in both the HOS-EC50 cells where it mediates MDR and the HOS-EC50.SR populations, whereas ABCG1 was only upregulated in the self-renewing drug resistant HOS-EC50.SR cells. Targeting ABCG1 and Pgp may eradicate the drug resistant self-renewing OST-CSCs, leading to improved outcomes for patients with OST.

Detecting ovarian cancer using extracellular vesicles: progress and possibilities.

Ovarian cancer (OC) is the deadliest gynecological malignancy. Most patients are diagnosed when they are already in the later stages of the disease. Earlier detection of OC dramatically improves the overall survival, but this is rarely achieved as there is a lack of clinically implemented biomarkers of early disease. Extracellular vesicles (EVs) are small cell-derived vesicles that have been extensively studied in recent years. They contribute to various aspects of cancer pathology, including tumor growth, angiogenesis and metastasis. EVs are released from all cell types and the macromolecular cargo they carry reflects the content of the cells from which they were derived. Cancer cells release EVs with altered cargo into biofluids, and so, they represent an excellent potential source of novel biomarkers for the disease. In this review, we describe the latest developments in EVs as potential biomarkers for earlier detection of OC. The field is still relatively young, but many studies have shown that EVs and the cargo they carry, including miRNAs and proteins, can be used to detect OC. They could also give insights into the stage of the disease and predict the likely therapeutic outcome. There remain many challenges to the use of EVs as biomarkers, but, through ongoing research and innovation in this exciting field, there is great potential for the development of diagnostic assays in the clinic that could improve patient outcome.

An overview of plant-based interventions to ameliorate arsenic toxicity.

The industrial and technological advancements in the world have also contributed to the rapid deterioration in the environment quality through introduction of obnoxious pollutants that threaten to destroy the subtle balance in the ecosystem. The environment contaminants cause severe adverse effects to humans, flora and fauna that are mostly irreversible. Chief among these toxicants is arsenic, a metalloid, which is considered among the most dangerous environmental toxins that leads to various diseases which affect the quality of life even when present in small quantities. Treatment of arsenic-mediated disorders still remains a challenge due to lack of effective options. Chelation therapy has been the most widely used method to detoxify arsenic. But this method is associated with deleterious effects leading various toxicities such as hepatotoxicity, neurotoxicity and other adverse effects. It has been discovered that indigenous drugs of plant origin display effective and progressive relief from arsenic-mediated toxicity without any side-effects. Further, these phytochemicals have also been found to aid the elimination of arsenic from the biological system and therefore can be more effective than conventional therapeutic agents in ameliorating arsenic-mediated toxicity. This review presents an overview of the toxic effects of arsenic and the therapeutic strategies that are available to mitigate the toxic effects with emphasis on chelation as well as protective and detoxifying activities of different phytochemicals and herbal drugs against arsenic. This information may serve as a primer in identifying novel prophylactic as well as therapeutic formulations against arsenic-induced toxicity.

A sixteen-week three-armed, randomized, controlled trial investigating clinical and biochemical effects of targeted alterations in dietary linoleic acid and n-3 EPA+DHA in adults with episodic migraine: Study protocol.

Migraine is a prevalent neurological disorder, affecting over 16% of adult women and 7% of adult men in the U.S., causing significant pain, disability, and medical expense, with incomplete benefits from conventional medical management. Migraine, as a chronic pain syndrome, provides a practical model for investigating the impact of dietary modifications in omega-3 (n-3) and omega-6 (n-6) fatty acids. This paper reports the protocol of a trial to assess whether targeted dietary modifications designed to increase n-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), with or without concurrent reduction in n-6 linoleic acid (LA), will alter nociceptive lipid mediators and mediate decreases in frequency and severity of migraine. This prospective, randomized, controlled trial in 153 male and female adult subjects, ages 18-99, with diagnosed and actively managed episodic migraine tests the efficacy, safety, and biochemical effects of targeted, controlled alterations in dietary omega-3 and omega-6 fatty acids. Participants are masked to diet hypotheses and all assessors are masked to treatment assignment. Following a four-week baseline period, participants with migraine headache frequency of 5-20 per month are randomized to one of three intensive dietary regimens for 16 additional weeks followed by a less intensive observation period. Dietary intervention arms include: 1) increased n-3 EPA+DHA with low n-6 linoleic acid (H3 L6); 2) increased n-3 EPA+DHA with usual US dietary intake of n-6 linoleic acid (H3 H6); and 3) usual US dietary content of n-3 and n-6 fatty acids (L3 H6). During the actual intervention, subjects receive content-specific study oils and foods sufficient for two meals and two snacks per day, as well as dietary counseling. Biochemical and clinical outcome measures are performed at intervals throughout this period. This randomized controlled trial is designed to determine whether targeted alterations in dietary n-3 and n-6 fatty acids can alter nociceptive lipid mediators in a manner that decreases headache pain and enhances quality of life and function in adults with frequent migraines. TRIAL REGISTRATION: NCT02012790.

Cisplatin induces the release of extracellular vesicles from ovarian cancer cells that can induce invasiveness and drug resistance in bystander cells.

Ovarian cancer has a poor overall survival that is partly caused by resistance to drugs such as cisplatin. Resistance can be acquired as a result of changes to the tumour or due to altered interactions within the tumour microenvironment. Extracellular vesicles (EVs), small lipid-bound vesicles that are loaded with macromolecular cargo and released by cells, are emerging as mediators of communication in the tumour microenvironment. We previously showed that EVs mediate the bystander effect, a phenomenon in which stressed cells can communicate with neighbouring naive cells leading to various effects including DNA damage; however, the role of EVs released following cisplatin treatment has not been tested. Here we show that treatment of cells with cisplatin led to the release of EVs that could induce invasion and increased resistance when taken up by bystander cells. This coincided with changes in p38 and JNK signalling, suggesting that these pathways may be involved in mediating the effects. We also show that EV uptake inhibitors could prevent this EV-mediated adaptive response and thus sensitize cells in vitro to the effects of cisplatin. Our results suggest that preventing pro-tumourigenic EV cross-talk during chemotherapy is a potential therapeutic target for improving outcome in ovarian cancer patients.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.

Construct validity of the Modified Gait Efficacy Scale in older females.

There are few well-validated tools that focus on the assessment of walking confidence in older adults. The main objective of this study was to assess construct validity of the 10-item Modified Gait Efficacy Scale (mGES) as a measure of walking confidence in older adults. Twenty-four older females completed the mGES, the 16-item Activities-specific Balance Confidence (ABC-16) scale, and the Senior Fitness Test (SFT). Construct validity of the mGES was evaluated by quantifying relationships between the mGES and the ABC-16 and the SFT, and by examining the ability of the mGES to discriminate between known groups (no/lower fear of falling versus higher fear of falling). There was a strong correlation between mGES and the ABC-16 scale (rs = 0.85; p < 0.001). The mGES was significantly associated with SFT components that required lower extremity strength, stepping aerobic endurance, and walking agility and dynamic balance (rs = 0.45 to 0.61; p < 0.05). Relationships between the mGES and number of arm curls in 30 s, chair sit and reach test, and back scratch test were weak (rs = 0.13-0.25; p > 0.05). Mean mGES score was 91.5% in a no/lower fear of falling group, while it was 81.4% in a higher fear group (p = 0.22). There was a trend toward a significant difference in the unstandardized residuals derived from regression of ranked mGES scores on ranked covariate (age and 8 foot up and go) scores, between the no/lower versus higher fear of falling group (p = 0.095). These results support construct validity of the mGES as a measure of gait self-efficacy in community-dwelling older females.

N-acetylgalactosamine glycans function in cancer cell adhesion to endothelial cells: A role for truncated O-glycans in metastatic mechanisms.

Failure in O-glycan chain extension exposing Tn antigen (GalNAc-O-Ser/Thr) is clinically associated with cancer metastasis. This study provides evidence of a functional role for aberrant GalNAc-glycans in cancer cell capture from blood flow and/or adhesion to endothelium. Adhesion of breast cancer cells to human umbilical vein endothelial cell monolayers was modelled under sweeping flow. Adhesion of metastatic, GalNAc glycan-rich, MCF7 and ZR 75 1 cells to endothelium increased over timepoints up to 1.5 hour, after which it plateaued. Adhesion was significantly inhibited (p < 0.001) when cell surface GalNAc-glycans were masked, an effect not seen in GalNAc glycan-poor, non-metastatic BT 474 cells. Masking irrelevant galactose- and mannose-glycans had no inhibitory effect. Imaging of cells post-adhesion over a 24 hour time course using confocal and scanning electron microscopy revealed that up to 6 hours post-adhesion, motile, rounded cancer cells featuring lamellipodia-like processes crawled on an intact endothelial monolayer. From 6-12 hours post-adhesion, cancer cells became stationary, adopted a smooth, circular flattened morphology, and endothelial cells retracted from around them leaving cleared zones in which the cancer cells proceeded to form colonies through cell division.

miRNAs and ovarian cancer: a miRiad of mechanisms to induce cisplatin drug resistance.

Ovarian cancer is the most aggressive gynecological cancer. One reason for the low 5-year survival rate of under 40% is that ovarian tumors usually acquire resistance to the platinum-based compounds used to treat them. Resistance to one such compound, cisplatin, can arise via numerous mechanisms that can be categorized as pre-, post-, on- or off-target. Pre-target mechanisms prevent accumulation of cisplatin in the cell, on-target mechanisms allow DNA damage to be repaired more efficiently, post-target mechanisms prevent the damage from inducing apoptosis and off-target mechanisms increase resistance via unrelated compensatory mechanisms. miRNAs are short non-coding RNAs that influence cellular function by repressing gene expression. Here we describe how miRNAs can induce cisplatin resistance in ovarian cancer cells via pre-, post-, on- and off-target mechanisms. A better understanding of how miRNAs feed into the mechanisms of drug resistance will inform the rational design of combination therapies for ovarian cancer.

Over-expression of miR-31 or loss of KCNMA1 leads to increased cisplatin resistance in ovarian cancer cells.

Ovarian cancers have a high mortality rate; this is in part due to resistance to the platinum-based compounds used in chemotherapy. In this paper, we assess the role of microRNA-31 in the development of chemoresistance to cisplatin. We used previous data from microarray experiments to identify potential microRNAs (miRNAs) involved in chemoresistance. The functional significance of these microRNAs was tested using miRNA mimics. We used RNA-seq to identify pathways and genes de-regulated in the resistant cell line and then determined their role using RNAi. Analysis of publically available datasets reveals the potential clinical significance. Our data show that miR-31 is increased, whilst potassium channel calcium activated large conductance subfamily M alpha, member 1 (KCNMA1), a subunit of calcium-regulated big potassium (BK) channels, is reduced in resistant ovarian cells. Over-expression of miR-31 increased resistance, as did knockdown of KCNMA1 or inhibition of BK channels. This suggests that these genes directly modulate cisplatin response. Our data also suggest that miR-31 represses KCNMA1 expression. Comparing the levels of miR-31 and KCNMA1 to cisplatin resistance in the NCI60 panel or chemoresistance in cohorts of ovarian cancer tumours reveals correlations that support a role for these genes in vitro and in vivo. Here we show that miR-31 and KCNMA1 are involved in mediating cisplatin resistance in ovarian cancer. Our data gives a new insight into the potential mechanisms to therapeutically target in cisplatin resistance common to ovarian cancer.