Resistance to chemotherapy in ovarian cancer is miRrored by the microRNA miR-433-dependent dysregulation of the cell cycle.

An understanding of the mechanisms underlying the development of resistance to chemotherapy treatment is a gateway to the introduction of novel therapies and improved outcomes for women presenting with ovarian cancer (OC). The desired apoptotic death post-chemotherapy depends on an intact and fully functioning cell cycle machinery.

In this study we demonstrate that stable expression of miR-433 renders OC cells more resistant to paclitaxel treatment. Interestingly, only cells with the highest miR-433 survived paclitaxel suggesting the possible role of miR-433 in cancer recurrence. Importantly, for the first time we demonstrate that miR 433 induces cellular senescence, exemplified by a flattened morphology, the downregulation of phosphorylated Retinoblastoma (p Rb) and increased β galactosidase activity. Surprisingly, miR 433 induced senescence was independent of two well recognised senescent drivers: p21 and p16. Further in silico analysis followed by in vitro experiments identified CKD6 as a novel miR-433 target gene possibly explaining the observed p21 and p16-independent induction of cellular senescence. Another in silico identified miR-433 target gene was CDC27, a protein involved in the regulation of the cell cycle during mitosis. We demonstrate that the overexpression of pre-miR-433 leads to the downregulation of CDC27 in vitro revealing a novel interaction between miR-433 and CDC27, an integral cell cycle regulating protein.

Interestingly, miR-433 expressing cells also demonstrated an ability to impact their tumour microenvironment. We show that miR-433 is present in exosomes released from miR-433 overexpressing and high miR-433 naïve cells. Moreover, growth condition media (GCM) harvested from cells with high miR-433 have higher levels of IL-6 and IL-8, two key cytokines involved in the senescence associated secretory phenotype (SASP). Importantly, GCM from miR-433-enriched cells repressed the growth of co-cultured cells with initial studies showing a GCM-dependent induction of chemoresistance.

In conclusion, data in this study highlights how the aberrant expression miR-433 contributes to chemoresistance in OC cells. We postulate that standard chemotherapy, particularly paclitaxel, used to treat women with OC may have an attenuated ability to kill cells harbouring increased levels of miR-433, allowing for a subsequent chemoresistant phenotype post-therapy.

A Candidate Gene Association Study Identifies DAPL1 as a Female-Specific Susceptibility Locus for Age-Related Macular Degeneration (AMD)

Age-related macular degeneration (AMD) is the leading cause of blindness among white caucasians over the age of 50 years with a prevalence rate expected to increase markedly with an anticipated increase in the life span of the world population. To further expand our knowledge of the genetic architecture of the disease, we pursued a candidate gene approach assessing 25 genes and a total of 109 variants. Of these, synonymous single nucleotide polymorphism (SNP) rs17810398 located in death-associated protein-like 1 (DAPL1) was found to be associated with AMD in a joint analysis of 3,229 cases and 2,835 controls from five studies [combined P ADJ = 1.15 × 10(-6), OR 1.332 (1.187-1.496)]. This association was characterized by a highly significant sex difference (P diff = 0.0032) in that it was clearly confined to females with genome-wide significance [P ADJ = 2.62 × 10(-8), OR 1.541 (1.324-1.796); males: P ADJ = 0.382, OR 1.084 (0.905-1.298)]. By targeted resequencing of risk and non-risk associated haplotypes in the DAPL1 locus, we identified additional potentially functional risk variants, namely a common 897-bp deletion and a SNP predicted to affect a putative binding site of an exonic splicing enhancer. We show that the risk haplotype correlates with a reduced retinal transcript level of two, less frequent, non-canonical DAPL1 isoforms. DAPL1 plays a role in epithelial differentiation and may be involved in apoptotic processes thereby suggesting a possible novel pathway in AMSaveD pathogenesis.

Utilisation of a novel ProteaseTag™ activity immunoassay for the specific measurement of neutrophil elastase in patients with Cystic Fibrosis

Neutrophil elastase (NE), a biomarker of infection and inflammation, correlates with the severity of several respiratory diseases including cystic fibrosis (CF) however, its detection and quantification in biological samples is confounded by a lack of robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex samples containing multiple proteolytic and hydrolytic enzymes, resulting in an over-estimation of the target protease. ELISA systems measure total protein levels which can be a mixture of latent, active and protease-inhibitor complexes. We have therefore developed a novel immunoassay (NE-Tag ELISA), incorporating an activity dependent ProteaseTag™ and a specific antibody step, which is selective and specific for the capture of active NE. The objective of this study was to clinically validate NE-Tag ELISA for the detection of active NE in sputum from CF patients. Sputum (n=45) was recovered from CF patients hospitalised for acute exacerbation. Sol was recovered and analysed for NE activity using the NE-Tag ELISA and two fluorogenic substrate-based assays [1. Suc-AAPV-AMC (Sigma) and 2. InnozymeTM Immunocapture assay (Calbiochem)]. NE activity between assays and with a range of clinical parameters was correlated.A highly significant correlation was shown between assays. NE activity (NE-Tag) further correlated appropriately with clinical parameters: inversely with FEV1 (p = 0.036) and positively with CRP (p = 0.035), neutrophils and total white cell counts (p < 0.001). The InnozymeTM assay showed similar correlations with the clinical parameters (with the exception of CRP). No correlations with any of the clinical parameters were observed when NE was measured using the standard fluorogenic substrate.

Regulating macrophage phenotype and function by retinal pigment epithelial cells

Purpose: We have shown previously that macrophages/microglia accumulate in the subretinal space and express CD68 and Arginase-1 in the aging eye. Subretinal macrophages are in close contact with retinal pigment epithelial (RPE) cells. We hypothesize that RPE cells may play an important role in regulating macrophage/microglial phenotype and function. The aim of this study was to investigate the effect of RPE cells on the phenotype and function of bone marrow–derived macrophages (BM-DMs).
Methods: BM-DM from C57BL/6J mice were cultured in DMEM supplemented with 20%L929 cell supernatant for 5 days. The phenotype of BM-DMs was confirmed by flow cytometry as CD11b+F4/80+. Primary RPE cells were cultured from C57BL/6J mice and confirmed by RPE65 and cytokeratin staining. BMDMs were co-cultured with different types of RPE cells (healthy, oxidized, and apoptotic RPE) and then isolated from the co-culture system for phenotypic and functional assays.
Results: Co-culture of BM-DMs with RPE cells results in a time-dependent down-regulation of MHC-II expression and the generation of CD11b+F4/80+Ly6G+ myeloid-derived suppressor cells (MDSC). qRT-PCR analysis showed that RPE-induced MDSCs expressed high levels of IL-6, IL-1β, and Arginase-1, but lower levels of IL-12p40 and TNF-a compared to naïve BM-DMs. The expression levels of iNOS, TGF-β and Ym1 did not differ 207 between naive BMDMs and RPE-induced MDSCs. Furthermore, functional studies showed that these cells had reduced phagocytic activity and lower ability to stimulate T cell activation and proliferation. When RPE cells were pre-treated with oxidized photoreceptor outer segments before co-culturing with BMDMs, the expression of IL-1β and IL-6 in BMDMs was increased whereas the expression of Arginase-1 was decreased. 
Conclusion: Our results suggest that healthy RPE cells can convert BMDMs into myeloid-derived suppressor cells under in vitro culture conditions, RPE-induced myeloid-derived suppressor cells are CD11b+F4/80+Ly6G+MHCIIlowIL6+IL1b+Arg-1+. The ability of RPE cells is reduced when suffering from oxidative insults.

Inflammatory and cytotoxic effects of acrolein, nicotine, acetylaldehyde and cigarette smoke extract on human nasal epithelial cells

Background: Cigarette smoke induces a pro-inflammatory response in airway epithelial cells but it is not clear which of the various chemicals contained within cigarette smoke (CS) should be regarded as predominantly responsible for these effects. We hypothesised that acrolein, nicotine and acetylaldehyde, important chemicals contained within volatile cigarette smoke in terms of inducing inflammation and causing addiction, have immunomodulatory effects in primary nasal epithelial cell cultures (PNECs).

Methods: PNECs from 19 healthy subjects were grown in submerged cultures and were incubated with acrolein, nicotine or acetylaldehyde prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide (PA LPS). Experiments were repeated using cigarette smoke extract (CSE) for comparison. IL-8 was measured by ELISA, activation of NF-κB by ELISA and Western blotting, and caspase-3 activity by Western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method.

Results: CSE was pro-inflammatory after a 24 h exposure and 42% of cells were apoptotic or necrotic after this exposure time. Acrolein was pro-inflammatory for the PNEC cultures (30 μM exposure for 4 h inducing a 2.0 fold increase in IL-8 release) and also increased IL-8 release after stimulation with PA LPS. In contrast, nicotine had anti-inflammatory properties (0.6 fold IL-8 release after 50 μM exposure to nicotine for 24 h), and acetylaldehyde was without effect. Acrolein and nicotine had cellular stimulatory and anti-inflammatory effects respectively, as determined by NF-κB activation. Both chemicals increased levels of cleaved caspase 3 and induced cell death.

Conclusions: Acrolein is pro-inflammatory and nicotine anti-inflammatory in PNEC cultures. CSE induces cell death predominantly by apoptotic mechanisms. 

The fate of chemoresistance in triple negative breast cancer (TNBC)

Background: Treatment options for women presenting with triple negative breast cancer (TNBC) are limited due to the lack of a therapeutic target and as a result, are managed with standard chemotherapy such as paclitaxel (Taxol®). Following chemotherapy, the ideal tumour response is apoptotic cell death. Post-chemotherapy, cells can maintain viability by undergoing viable cellular responses such as cellular senescence, generating secretomes which can directly enhance the malignant phenotype. 
Scope of Review: How tumour cells retain viability in response to chemotherapeutic engagement is discussed. In addition we discuss the implications of this retained tumour cell viability in the context of the development of recurrent and metastatic TNBC disease. Current adjuvant and neo-adjuvant treatments available and the novel potential therapies that are being researched are also reviewed. 
Major conclusions: Cellular senescence and cytoprotective autophagy are potential mechanisms of chemoresistance in TNBC. These two non-apoptotic outcomes in response to chemotherapy are inextricably linked and are neglected outcomes of investigation in the chemotherapeutic arena. Cellular fate assessments may therefore have the potential to predict TNBC patient outcome. 
General Significance: Focusing on the fact that cancer cells can bypass the desired cellular apoptotic response to chemotherapy through cellular senescence and cytoprotective autophagy will highlight the importance of targeting non-apoptotic survival pathways to enhance chemotherapeutic efficacy

Abstract 1905: Defining a therapeutic classification of breast cancer by actionable targets

Breast cancer remains a frequent cause of female cancer death despite the great strides in elucidation of biological subtypes and their reported clinical and prognostic significance. We have defined a general cohort of breast cancers in terms of putative actionable targets, involving growth and proliferative factors, the cell cycle, and apoptotic pathways, both as single biomarkers across a general cohort and within intrinsic molecular subtypes.

We identified 293 patients treated with adjuvant chemotherapy. Additional hormonal therapy and trastuzumab was administered depending on hormonal and HER2 status respectively. We performed immunohistochemistry for ER, PR, HER2, MM1, CK5/6, p53, TOP2A, EGFR, IGF1R, PTEN, p-mTOR and e-cadherin. The cohort was classified into luminal (62%) and non-luminal (38%) tumors as well as luminal A (27%), luminal B HER2 negative (22%) and positive (12%), HER2 enriched (14%) and triple negative (25%). Patients with luminal tumors and co-overexpression of TOP2A or IGF1R loss displayed worse overall survival (p=0.0251 and p=0.0008 respectively). Non-luminal tumors had much greater heterogeneous expression profiles with no individual markers of prognostic significance. Non-luminal tumors were characterised by EGFR and TOP2A overexpression, IGF1R, PTEN and p-mTOR negativity and extreme p53 expression.

Our results indicate that only a minority of intrinsic subtype tumors purely express single novel actionable targets. This lack of pure biomarker expression is particular prevalent in the triple negative subgroup and may allude to the mechanism of targeted therapy inaction and myriad disappointing trial results. Utilising a combinatorial biomarker approach may enhance studies of targeted therapies providing additional information during design and patient selection while also helping decipher negative trial results.

Intracellular survival of Burkholderia cepacia complex in phagocytic cells

Burkholderia cepacia complex (Bcc) species are a group of Gram-negative opportunistic pathogens that infect the airways of cystic fibrosis patients, and occasionally they infect other immunocompromised patients. Bcc bacteria display high-level multidrug resistance, and chronically persist in the infected host while eliciting robust inflammatory responses. Studies using macrophages, neutrophils and dendritic cells, combined with advances to genetically manipulate these bacteria have increased our understanding of the molecular mechanisms of virulence in these pathogens and the molecular details of cell-host responses triggering inflammation. This article discusses our current view of the intracellular survival of B. cenocepacia within macrophages.

The Role of Serine Proteases and Antiproteases in the Cystic Fibrosis Lung

Cystic fibrosis (CF) lung disease is an inherited condition with an incidence rate of approximately 1 in 2500 new born babies. CF is characterized as chronic infection of the lung which leads to inflammation of the airway. Sputum from CF patients contains elevated levels of neutrophils and subsequently elevated levels of neutrophil serine proteases. In a healthy individual these proteases aid in the phagocytic process by degrading microbial peptides and are kept in homeostatic balance by cognate antiproteases. Due to the heavy neutrophil burden associated with CF the high concentration of neutrophil derived proteases overwhelms cognate antiproteases. The general effects of this protease/antiprotease imbalance are impaired mucus clearance, increased and self-perpetuating inflammation, and impaired immune responses and tissue. To restore this balance antiproteases have been suggested as potential therapeutics or therapeutic targets. As such a number of both endogenous and synthetic antiproteases have been trialed with mixed success as therapeutics for CF lung disease.

Evidence supporting a role for N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine accumulation in Muller glia dysfunction and death in diabetic retinopathy

Purpose: Recent evidence suggests that neuroglial dysfunction and degeneration contributes to the etiology and progression of diabetic retinopathy. Advanced lipoxidation end products (ALEs) have been implicated in the pathology of various diseases, including diabetes and several neurodegenerative disorders. The purpose of the present study was to investigate the possible link between the accumulation of ALEs and neuroretinal changes in diabetic retinopathy.

Methods: Retinal sections obtained from diabetic rats and age-matched controls were processed for immunohistochemistry using antibodies against several well defined ALEs. In vitro experiments were also performed using a human Muller (Moorfields/Institute of Ophthalmology-Muller 1 [ MIO-M1]) glia cell line. Western blot analysis was used to measure the accumulation of the acrolein-derived ALE adduct N epsilon-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) in Muller cells preincubated with FDP-lysine-modified human serum albumin (FDP-lysine-HSA). Responses of Muller cells to FDP-lysine accumulation were investigated by analyzing changes in the protein expression of heme oxygenase-1 (HO-1), glial fibrillary acidic protein (GFAP), and the inwardly rectifying potassium channel Kir4.1. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) were determined by reverse transcriptase PCR (RT-PCR). Apoptotic cell death was evaluated by fluorescence-activated cell sorting (FACS) analysis after staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide.

Results: No significant differences in the levels of malondialdehyde-, 4-hydroxy-2-nonenal-, and 4-hydroxyhexenal-derived ALEs were evident between control and diabetic retinas after 4 months of diabetes. By contrast, FDP-lysine immunoreactivity was markedly increased in the Muller glia of diabetic rats. Time-course studies revealed that FDP-lysine initially accumulated within Muller glial end feet after only a few months of diabetes and thereafter spread distally throughout their inner radial processes. Exposure of human Muller glia to FDP-lysine-HSA led to a concentration-dependent accumulation of FDP-lysine-modified proteins across a broad molecular mass range. FDP-lysine accumulation was associated with the induction of HO-1, no change in GFAP, a decrease in protein levels of the potassium channel subunit Kir4.1, and upregulation of transcripts for VEGF, IL-6, and TNF-alpha. Incubation of Muller glia with FDP-lysine-HSA also caused apoptosis at high concentrations.

Conclusions: Collectively, these data strongly suggest that FDP-lysine accumulation could be a major factor contributing to the Muller glial abnormalities occurring in the early stages of diabetic retinopathy.

Innate immune activation in neovascular Age-related Macular Degeneration

Purpose: To compare white blood cell populations from persons with neovascular age-related macular degeneration (nAMD) with that of age-matched controls.

Methods: Immunophenotyping for white blood cell populations (including CD14++CD16-, CD14++CD16+ and CD14+CD16++ monocytes, CD4 and CD8 T-lymphocytes, CD56 natural killer cells, CD19 B-lymphocytes and CD16+HLA-DR- neutrophils), chemokine receptor expression analysis (CX3CR1 and CCR2) as well as cell activation analysis (MHC-II, HLA-DR, CD62L, STAT3) was performed using samples of peripheral blood from nAMD patients and age- and gender-matched controls.

Results: The percentage of CD4+ T cells was significantly reduced while the percentage of CD11b+ cells and CD16+HLA-DR- neutrophils was significantly increased in nAMD patients compared to controls. The percentage of classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocytes was similar between nAMD patients and controls, however there was a significant increase of CX3CR1 on the intermediate monocyte subset and on CD16+HLA-DR- neutrophils in nAMD compared to controls. HLA-DR was significantly increased in all monocyte subsets in nAMD compared to controls. Activation of Signal Transducer and Activator of Transcription 3 (STAT3) was significantly increased in nAMD patients compared to controls following stimulation with IL6.

Conclusions: Our results suggest an increased activation of the innate immune system in patients with nAMD. A better understanding of the role of the innate immune system in the pathogenesis of nAMD may help identify novel biomarkers and thus development of improved therapeutic strategies.

Molecular mechanisms of virulence of Burkholderia cepacia complex bacteria: Survival in Phagocytic Cells

Burkholderia cepacia complex (Bcc) species are a group of Gram-negative opportunistic pathogens that chronically infect the airways of cystic fibrosis patients, but they can also infect patients with various types of immunosuppressive disorders. Bcc members are multidrug resistant bacteria that have the ability to persist in the infected host and also elicit robust inflammatory responses. Studies using macrophages, neutrophils and dendritic cells, combined with dramatic advances in the ability to genetically manipulate these microorganisms have contributed to increase our understanding of the molecular mechanisms of virulence in these pathogens and the molecular details of the cell host responses triggering inflammation. This chapter reviews our understanding of the pathogenic mechanisms used by Bcc to establish an intracellular niche in phagocytic cells and modulate host cell responses that ultimately end up in cell death and a proinflammatory response.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce cell cycle arrest and apoptosis in prostate cancer cells

Advanced hormone-refractory prostate cancer is associated with poor prognosis and limited treatment options. Members of the pyrrolo-1,5-benzoxazepine (PBOX) family of compounds exhibit anti-cancer properties in cancer cell lines (including multi-drug resistant cells), ex vivo patient samples and in vivo mouse tumour models with minimal toxicity to normal cells. Recently, they have also been found to possess anti-angiogenic properties in vitro. However, both the apoptotic pathways and the overall extent of the apoptotic response induced by PBOX compounds tend to be cell-type specific. Since the effect of the PBOX compounds on prostate cancer has not yet been elucidated, the purpose of this study was to investigate if PBOX compounds induce anti-proliferative effects on hormone-refractory prostate cancer cells. We examined the effect of two representative PBOX compounds, PBOX-6 and PBOX-15, on the androgen-independent human prostate adenocarcinoma cell line, PC3. PBOX-6 and -15 displayed anti-proliferative effects on PC3 cells, mediated initially through a sustained G2/M arrest. G2/M arrest, illustrated as DNA tetraploidy, was accompanied by microtubule depolymerisation and phosphorylation of anti-apoptotic proteins Bcl-2 and Bcl-xL and the mitotic spindle checkpoint protein BubR1. Phosphorylation of BubR1 is indicative of an active mitotic checkpoint and results in maintenance of cell cycle arrest. G2/M arrest was followed by apoptosis illustrated by DNA hypoploidy and PARP cleavage and was accompanied by degradation of BubR1, Bcl-2 and Bcl-xL. Furthermore, sequential treatment with the CDK1-inhibitor, flavopiridol, synergistically enhanced PBOX-induced apoptosis. In summary, this in vitro study indicates that PBOX compounds may be useful alone or in combination with other agents in the treatment of hormone-refractory prostate cancer.

The vascular targeting agent combretastatin-A4 and a novel cis-Restricted {beta}-Lactam Analogue, CA-432, induce apoptosis in human chronic myeloid leukemia cells and ex vivo patient samples including those displaying multidrug resistance

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (β-lactam) ring were designed and synthesized with the objective to prevent cis -trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected β-lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead β-lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G(2)M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-x(L) preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and β-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized.

Sequential treatment with flavopiridol synergistically enhances pyrrolo-1,5-benzoxazepine-induced apoptosis in human chronic myeloid leukaemia cells including those resistant to imatinib treatment

The Bcr-Abl kinase inhibitor, imatinib mesylate, is the front line treatment for chronic myeloid leukaemia (CML), but the emergence of imatinib resistance has led to the search for alternative drug treatments and the examination of combination therapies to overcome imatinib resistance. The pro-apoptotic PBOX compounds are a recently developed novel series of microtubule targeting agents (MTAs) that depolymerise tubulin. Recent data demonstrating enhanced MTA-induced tumour cell apoptosis upon combination with the cyclin dependent kinase (CDK)-1 inhibitor flavopiridol prompted us to examine whether this compound could similarly enhance the effect of the PBOX compounds. We thus characterised the apoptotic and cell cycle events associated with combination therapy of the PBOX compounds and flavopiridol and results showed a sequence dependent, synergistic enhancement of apoptosis in CML cells including those expressing the imatinib-resistant T315I mutant. Flavopiridol reduced the number of polyploid cells formed in response to PBOX treatment but only to a small extent, suggesting that inhibition of endoreplication was unlikely to play a major role in the mechanism by which flavopiridol synergistically enhanced PBOX-induced apoptosis. The addition of flavopiridol following PBOX-6 treatment did however result in an accelerated exit from the G2/M transition accompanied by an enhanced downregulation and deactivation of the CDK1/cyclin B1 complex and an enhanced degradation of the inhibitor of apoptosis protein (IAP) survivin. In conclusion, results from this study highlight the potential of these novel series of PBOX compounds, alone or in sequential combination with flavopiridol, as an effective therapy against CML.