Paula Cunnea

The effects of blood–brain barrier disruption on glial cell function in multiple sclerosis

Dysfunction of the BBB (blood-brain barrier) is a major hallmark of MS (multiple sclerosis). Studies in our laboratories over the last decade have shown that increased BBB permeability is associated with decreased expression of TJ (tight junction) proteins in brain capillary endothelial cells. Results have revealed that TJ abnormalities were most common in active lesions (42% of vessels affected), but were also present in inactive lesions (23%) and in MS normal-appearing white matter (13%). Importantly, TJ abnormality was also positively associated with leakage of the serum protein fibrinogen which has recently been shown to be an activator of microglia. TJ abnormality and the resultant vascular permeability in both lesional and non-lesional white matter may impair tissue homoeostasis, which may have effects on disease progression, repair mechanisms and drug delivery.

Expression profiles of endoplasmic reticulum stress-related molecules in demyelinating lesions and multiple sclerosis

Background: Increasing evidence associates the endoplasmic reticulum (ER) stress signalling pathway as a potential treatment target in multiple sclerosis (MS). Objective: To establish the expression profile of markers of ER stress both in demyelinating biopsy specimens and microdissected lesions in human post-mortem MS tissue. Methods: Immunohistochemical detection of C/EBP homologous protein (CHOP), immunoglobulin heavy chain binding protein (BiP), and hypoxia marker antigen D-110 in biopsies from three patients with MS primary or secondary progressive, three patients with clinically isolated syndrome, and one patient with lesional epilepsy was carried out. Laser capture microdissection of normal, perilesion and lesion tissue from post-mortem MS tissue and non-diseased control tissue was performed, followed by real-time PCR to detect ER stress genes. Results: In biopsy specimens, increased expression of the ER and hypoxic stress molecules in a range of cell types in most of the actively demyelinating lesions and perilesions was detected. Real-time PCR analysis demonstrated statistically significant elevated expression of the ER stress genes in normal-appearing white matter relative to control white matter. Moreover, significantly increased expression of CHOP was detected in the perilesion of active plaques (p < 0.01). Conclusions: Our results, showing detection of elevated expression of ER stress molecules in lesional tissue, offer compelling evidence for further investigation of the ER stress signalling pathway as a potential therapeutic target for the treatment of MS.

The role of interleukin-8 (IL-8) and IL-8 receptors in platinum response in high grade serous ovarian carcinoma.

Platinum based drugs are the cornerstone of chemotherapy for ovarian cancer, however the development of chemoresistance hinders its success. IL-8 is involved in regulating several pro-survival pathways in cancer. We studied the expression of IL-8 and IL-8 receptors in platinum sensitive and resistant cell lines. Using qRT-PCR and immunohistochemistry, both platinum sensitive (PEA1, PEO14) and resistant (PEA2, PEO23) show increased expression of IL-8 and IL-8 receptors. IL-8RA shows nuclear and cytoplasmic expression, whilst IL-8RB is present solely in the cytoplasm. Knockdown of IL-8 increased sensitivity to cisplatin in platinum sensitive and reversed platinum resistance in resistant cell lines, decreased the expression of anti-apoptotic Bcl-2 and decreased inhibitory phosphorylation of pro-apoptotic Bad. IL-8 receptor antagonist treatment also enhanced platinum sensitivity. Nuclear localisation of IL-8RA was only detected in platinum resistant tumours. Inhibition of IL-8 signalling can enhance response in platinum sensitive and resistant disease. Nuclear IL-8RA may have potential as a biomarker of resistant disease.

A putative biomarker signature for clinically effective AKT inhibition: correlation of in vitro , in vivo and clinical data identifies the importance of modulation of the mTORC1 pathway

Our identification of dysregulation of the AKT pathway in ovarian cancer as a platinum resistance specific event led to a comprehensive analysis of in vitro, in vivo and clinical behaviour of the AKT inhibitor GSK2141795. Proteomic biomarker signatures correlating with effects of GSK2141795 were developed using in vitro and in vivo models, well characterised for related molecular, phenotypic and imaging endpoints. Signatures were validated in temporally paired biopsies from patients treated with GSK2141795 in a clinical study. GSK2141795 caused growth-arrest as single agent in vitro, enhanced cisplatin-induced apoptosis in vitro and reduced tumour volume in combination with platinum in vivo. GSK2141795 treatment in vitro and in vivo resulted in ~50-90% decrease in phospho-PRAS40 and 20-80% decrease in fluoro-deoxyglucose (FDG) uptake. Proteomic analysis of GSK2141795 in vitro and in vivo identified a signature of pathway inhibition including changes in AKT and p38 phosphorylation and total Bim, IGF1R, AR and YB1 levels. In patient biopsies, prior to treatment with GSK2141795 in a phase 1 clinical trial, this signature was predictive of post-treatment changes in the response marker CA125. Development of this signature represents an opportunity to demonstrate the clinical importance of AKT inhibition for re-sensitisation of platinum resistant ovarian cancer to platinum.

Modeling platinum sensitive and resistant high-grade serous ovarian cancer: development and applications of experimental systems

High-grade serous ovarian cancer remains the most common sub-type of ovarian cancer and, characterized by high degrees of genomic instability and heterogeneity, is typified by a transition from early response to acquired resistance to platinum-based chemotherapy. Conventional models for the study of ovarian cancer have been largely limited to a set of relatively poorly characterized immortalized cell lines and recent studies have called into question the validity of some of these as reliable models. Here, we review new approaches and models systems that take into account advances in our understanding of ovarian cancer biology and advances in the technology available for their generation and study. We discuss primary cell models, 2D, 3D, and organotypic models, and “paired” sample approaches that capture the evolution of chemotherapy failure within single cases. We also overview new methods for non-invasive collection of representative tumor material from blood samples. Adoption of such methods and models will improve the quality and clinical relevance of ovarian cancer research.

The tumour suppressor OPCML promotes AXL inactivation by the phosphatase PTPRG in ovarian cancer

In ovarian cancer, the prometastatic RTK AXL promotes motility, invasion and poor prognosis. Here, we show that reduced survival caused by AXL overexpression can be mitigated by the expression of the GPI‐anchored tumour suppressor OPCML. Further, we demonstrate that AXL directly interacts with OPCML, preferentially so when AXL is activated by its ligand Gas6. As a consequence, AXL accumulates in cholesterol‐rich lipid domains, where OPCML resides. Here, phospho‐AXL is brought in proximity to the lipid domain‐restricted phosphatase PTPRG, which de‐phosphorylates the RTK/ligand complex. This prevents AXL‐mediated transactivation of other RTKs (cMET and EGFR), thereby inhibiting sustained phospho‐ERK signalling, induction of the EMT transcription factor Slug, cell migration and invasion. From a translational perspective, we show that OPCML enhances the effect of the phase II AXL inhibitor R428 in vitro and in vivo. We therefore identify a novel mechanism by which two spatially restricted tumour suppressors, OPCML and PTPRG, coordinate to repress AXL‐dependent oncogenic signalling.

A method for voltage measurements of cancerous vs non-cancerous omentum.

This paper presents and elaborates upon the practicalities of a method which enables the recording of voltage measurements from omental tissue in patients with advanced ovarian cancer. The key components of the proposed low-cost experimental setup are a tungsten electrode, a Ag/AgCl reference electrode and an instrumentation amplifier. Intriguingly, potential difference recordings between cancerous omentum and tissue culture media and between non-cancerous omentum and media, differ for tissue samples coming from the same patient. Further studies are warranted to assess the potential prognostic value of voltage measurements in cancerous tissue.

Clinical value of bioelectrical properties of cancerous tissue in advanced epithelial ovarian cancer patients

Currently, there are no valid pre-operatively established biomarkers or algorithms that can accurately predict surgical and clinical outcome for patients with advanced epithelial ovarian cancer (EOC). In this study, we suggest that profiling of tumour parameters such as bioelectrical-potential and metabolites, detectable by electronic sensors, could facilitate the future development of devices to better monitor disease and predict surgical and treatment outcomes. Biopotential was recorded, using a potentiometric measurement system, in ex vivo paired non-cancerous and cancerous omental tissues from advanced stage EOC (n = 36), and lysates collected for metabolite measurement by microdialysis. Consistently different biopotential values were detected in cancerous tissue versus non-cancerous tissue across all cases (p < 0.001). High tumour biopotential levels correlated with advanced tumour stage (p = 0.048) and tumour load, and negatively correlated with stroma. Within our EOC cohort and specifically the high-grade serous subtype, low biopotential levels associated with poorer progression-free survival (p = 0.0179, p = 0.0143 respectively). Changes in biopotential levels significantly correlated with common apoptosis related pathways. Lactate and glucose levels measured in paired tissues showed significantly higher lactate/glucose ratio in tissues with low biopotential (p < 0.01, n = 12). Our study proposes the feasibility of biopotential and metabolite monitoring as a biomarker modality profiling EOC to predict surgical and clinical outcomes.

The passive biomechanics of human pelvic collecting lymphatic vessels

The lymphatic system has a major significance in the metastatic pathways in women’s cancers. Lymphatic pumping depends on both extrinsic and intrinsic mechanisms, and the mechanical behavior of lymphatic vessels regulates the function of the system. However, data on the mechanical properties and function of human lymphatics are lacking. Our aim is to characterize, for the first time, the passive biomechanical behavior of human collecting lymphatic vessels removed at pelvic lymph node dissection during primary debulking surgeries for epithelial ovarian cancer. Isolated vessels were cannulated and then pressurized at varying levels of applied axial stretch in a calcium-free Krebs buffer. Pressurized vessels were then imaged using multi-photon microscopy for collagen-elastin structural composition and fiber orientation. Both pressure-diameter and force-elongation responses were highly nonlinear, and axial stretching of the vessel served to decrease diameter at constant pressure. Pressure-diameter behavior for the human vessels is very similar to data from rat mesenteric vessels, though the human vessels were approximately 10× larger than those from rats. Multiphoton microscopy revealed the vessels to be composed of an inner layer of elastin with an outer layer of aligned collagen fibers. This is the first study that successfully described the passive biomechanical response and composition of human lymphatic vessels in patients with ovarian cancer. Future work should expand on this knowledge base with investigations of vessels from other anatomical locations, contractile behavior, and the implications on metastatic cell transport.

Abstract A47: DNA-PKcs is amplified in high-grade serous ovarian cancer (HGSC), correlates with poor outcome and drives resistance to platinum therapy via the AKT signaling pathway

High grade serous ovarian cancer is typified by p53 mutation, high degrees of genomic instability and the development of chemo-resistance. Genomic translocations result from incorrectly repaired DNA double strand breaks (DSBs). DNA-PKcs is a central catalytic component of the error prone non-homologous end joining (NHEJ) DSB repair mechanism. We report here that DNA-PKcs is frequently amplified in copy number in HGSC and that amplification correlates with higher DNA-PKcs gene expression and poorer patient outcome (PFS/OS) using both in-house and publicly available datasets (TCGA). Targeting DNA-PKcs, pharmacologically or by RNAi, enhances apoptosis in response to platinum treatment in platinum resistant cell lines and primary models. Furthermore we report that inhibition of DNA-PK restores response to cisplatin in chemoresistant ovarian cancer cells in vivo. SKOV-3 tumor xenografts were implanted subcutaneously into Balb/c Nu/Nu mice and treated with DNA-PKcs inhibitor NU7441 for 2 weeks alone or in combination with cisplatin. DNA-PK inhibition or cisplatin treatment alone were ineffective however in combination they decreased tumor growth at 14 days by 90%, relative to platinum only treatment. On investigating the mechanism of DNA-PKcs mediated chemoresistance we revealed that, in response to DNA damage, DNA-PKcs phosphorylates AKT on serine residue 473 in the nucleus of platinum resistant ovarian tumor cells, but not sensitive cells from the same patient. Phosphorylation at AKT threonine 308 is unaffected and DNA-PK inhibition does not interfere with insulin-mediated phosphorylation of AKT S473. DNA-PK mediated AKT activation in chemoresistant cells results in inhibitory phosphorylation of the pro-apoptotic protein BAD and stabilisation of the anti-apoptotic Bcl-2 protein resulting in the observed attenuation of apoptotic response to platinum treatment. DNA-PK inhibition is an attractive therapeutic strategy for resensitising resistant tumors to platinum based therapy, directly linking the DNA damage caused by platinum to the pro-survival AKT pathway, without interfering with normal physiological roles of AKT. Citation Format: Curry E, Cheraghchi-Bashi-Astaneh A, Chen M, Cunnea P, De Sousa C, Maginn E, Dai Y, Liu E, Wasan H, Mills G, Bowtell D, Gabra H & Stronach EA. DNA-PKcs is amplified in high grade serous ovarian cancer (HGSC), correlates with poor outcome and drives resistance to platinum therapy via the AKT signaling pathway [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS20.