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// Christine C. Dobson 1, 2 , Thet Naing 1 , Shawn T. Beug 1 , Mame D. Faye 1 , Janelle Chabot 1 , Martin St-Jean 1 , Danielle E. Walker 1 , Eric C. LaCasse 1 , David F. Stojdl 1, 3 , Robert G. Korneluk 1, 3 , Martin Holcik 1, 4 1 Molecular Biomedicine Program, Apoptosis Research Centre, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, ON, Canada 2 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada 3 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada 4 Department of Pediatrics, University of Ottawa, Ottawa, ON, Canada Correspondence to: Martin Holcik, email: martin@arc.cheo.ca Keywords: rhabdomyosarcoma, Smac mimetic, oncolytic virus, immunotherapy Received: June 20, 2016 Accepted: November 23, 2016 Published: December 10, 2016 ABSTRACT Rhabdomyosarcoma (RMS), a neoplasm characterized by undifferentiated myoblasts, is the most common soft tissue tumour in children. Therapeutic resistance is common in RMS and is often caused by acquired defects in the cellular apoptotic program. Smac mimetic compounds (SMCs) are a novel class of inhibitor of apoptosis (IAP) antagonists that are currently under clinical development as cancer therapeutics. We previously reported that cIAP1 is overexpressed in human primary RMS tumours and in patient-derived RMS cell lines where it drives resistance to apoptosis. In this study, we investigated whether inflammatory cytokine production triggered by activators of innate immunity synergizes with LCL161 to induce bystander killing of RMS cells in vitro and in vivo . Indeed, we show that innate immune stimuli (oncolytic virus (VSVΔ51-GFP), interferon γ (IFNγ), and tumour necrosis factor-like weak inducer of apoptosis (TWEAK)) combine with SMCs in vitro to reduce cell viability in the Kym-1 RMS cancer cell line. Other human RMS cell lines (RH36, RH41, RD, RH18, RH28, and RH30) and the murine RMS cell line 76-9 are resistant to treatment with LCL161 alone or in combination with immune stimulants in in vitro cell viability assays. In contrast, we report that the combination of LCL161 and VSVΔ51-GFP reduces tumour volume and prolongs survival in a 76-9 syngeneic murine model. Our results support further exploration of the combined use of IAP antagonists and innate immune stimuli as a therapeutic approach for RMS cancers.

Rhabdomyosarcoma (RMS), a malignant neoplasm of presumed mesenchymal origin, is the most common soft tissue cancer of childhood. Despite aggressive treatment, resistance to current therapies remains a challenge. The success of most cytotoxic chemotherapies requires intact programmed cell death (apoptosis) pathways. Defects in the cellular apoptotic program play a key role in the pathogenesis of RMS and contribute to chemotherapeutic resistance to current treatments. Targeting and engaging apoptotic pathways using small-molecule IAP antagonists, death-inducing ligands, reestablishing pannexin channel expression and activity, immunotherapies, or a combination of these approaches is expected to improve outcomes in RMS patients. There is a clear need to better understand the molecular basis of apoptotic resistance in RMS, which may provide an opportunity to identify the patients most likely to benefit from targeted treatments, and for the discovery of novel therapies.

// Urszula Liwak-Muir 1 , Christine C. Dobson 1 , Thet Naing 1 , Quinlan Wylie 1 , Lucia Chehade 1 , Stephen D. Baird 1 , Pranesh K. Chakraborty 2,3 and Martin Holcik 1,2 1 Molecular Biomedicine Program, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, ON, Canada 2 Department of Pediatrics, University of Ottawa, Ottawa, ON, Canada 3 Newborn Screening Ontario, Children’s Hospital of Eastern Ontario, University of Ottawa, Ottawa, ON, Canada Correspondence to: Martin Holcik, email: // Keywords : tumour supressor, miRNA, kinase Received : September 14, 2015 Accepted : November 16, 2015 Published : November 22, 2015 Abstract Programmed cell death 4 (PDCD4) is a tumour suppressor implicated in cancer development and progression and was recently identified as a repressor of cap-independent translation of specific genes involved in the regulation of apoptosis. We show that the RNA-binding protein HuR binds to the PDCD4 3’UTR to protect it from miR-21-induced silencing. However, following H 2 O 2 treatment, PDCD4 mRNA is degraded via miR-21 binding. Importantly, we identify HuR as a novel substrate of the ERK8 kinase pathway in response to H 2 O 2 treatment. We show that phosphorylation of HuR by ERK8 prevents it from binding to PDCD4 mRNA and allows miR-21-mediated degradation of PDCD4.

Maternal ethanol consumption during pregnancy can produce a range of teratogenic outcomes in offspring. The mechanism of ethanol teratogenicity is multi-faceted, but may involve alterations in insulin and insulin-like growth factor (IGF) signaling pathways. These pathways are not only important for metabolism, but are also critically involved in neuronal survival and plasticity, and they can be altered by chronic prenatal ethanol exposure (CPEE). The objective of this study was to test the hypothesis that CPEE alters expression of insulin and IGF signaling molecules in the prefrontal cortex and liver of adult guinea pig offspring. Pregnant Dunkin-Hartley-strain guinea pigs received ethanol (4 g/kg maternal body weight/day) or isocaloric-sucrose/pair-feeding (nutritional control) throughout gestation. Fasting blood glucose concentration was measured in male and female offspring at postnatal day 150-200, followed by euthanasia, collection of prefrontal cortex and liver, and RNA extraction. IGF-1, IGF-1 receptor (IGF-1R), IGF-2, IGF-2 receptor (IGF-2R), insulin receptor substrate (IRS)-1, IRS-2, and insulin receptor (INSR) mRNA expression levels were measured in tissues using quantitative real-time PCR. The mean maternal blood ethanol concentration was 281 ± 15 mg/dL at 1 h after the second divided dose of ethanol on GD 57. CPEE resulted in increased liver weight in adult offspring, but produced no difference in fasting blood glucose concentration compared with nutritional control. In the liver, CPEE decreased mRNA expression of IGF-1, IGF-1R, and IGF-2, and increased IRS-2 mRNA expression in male offspring only compared with nutritional control. Female CPEE offspring had decreased INSR hepatic mRNA expression compared with male CPEE offspring. In the prefrontal cortex, IRS-2 mRNA expression was increased in CPEE offspring compared with nutritional control. The data demonstrate that CPEE alters both central and peripheral expression of insulin and IGF signaling molecules at the mRNA level, which may be related to metabolic dysregulation in adult offspring. Furthermore, altered insulin and IGF signaling may be a mechanism of ethanol neurobehavioral teratogenicity.

Maternal-fetal signaling is critical for optimal fetal development and postnatal outcomes. Chronic ethanol exposure alters programming of the fetal hypothalamic-pituitary-adrenal (HPA) axis, resulting in a myriad of neurochemical and behavioral alterations in postnatal life. Based on a recent study which showed that human intra-partum fetal stress increased fetal secretion of corticosterone, the non-dominant glucocorticoid, this investigation tested the hypothesis that an established model of HPA axis programming, chronic maternal ethanol administration to the pregnant guinea pig, would result in preferential elevation of corticosterone, which is also the non-dominant glucocorticoid. Starting on gestational day (GD) 2, guinea pigs received oral administration of ethanol (4 g/kg maternal body weight/day) or isocaloric-sucrose/pair-feeding. Each treatment was administered daily and continued until GD 45, 55, or 65 (approximately 3 days pre-term), when pregnant animals were euthanized and fetuses delivered by Caesarean section. Maternal and fetal plasma samples were collected. After sample preparation (protein precipitation and C-18 solid phase extraction), plasma cortisol and corticosterone concentrations were determined simultaneously by liquid chromatography coupled to tandem mass spectrometry. As predicted, chronic ethanol exposure increased both fetal and maternal plasma corticosterone concentration in late gestation. In contrast, plasma cortisol did not differ across maternal treatments in maternal or fetal samples. The plasma concentration of both maternal glucocorticoids increased with gestational age. Thus, corticosterone, the non-dominant glucocorticoid, but not cortisol, was elevated by chronic ethanol exposure, which may have effects on HPA function in later life.

Ethanol consumption during pregnancy can produce a variety of teratogenic effects in offspring, termed Fetal Alcohol Spectrum Disorders (FASD). The most debilitating and permanent consequence of chronic prenatal ethanol exposure (CPEE) is neurobehavioral teratogenicity, which often manifests as cognitive and behavioral impairments, including deficits in spatial learning and memory. This study tested the hypothesis that a modified dry-land version of the multi-choice Biel-maze task is more sensitive than the rewarded-alternation Y-maze task for the determination of spatial learning and memory deficits of ethanol teratogenicity. Pregnant guinea pigs received ethanol (4 g/kg maternal body weight/day) or isocaloric-sucrose/pair-feeding (control) for 5days/week throughout gestation. CPEE resulted in ethanol neurobehavioral teratogenicity in offspring, as demonstrated by increased spontaneous locomotor activity at postnatal day (PD) 10 and decreased brain weight at euthanasia (PD 150-200). On PD 21, offspring were randomly assigned to one of two tasks to assess spatial learning and memory performance: a dry-land version of the Biel maze or a rewarded-alternation Y-maze. Animals were habituated to the environment of their assigned task and performance of each CPEE or control offspring was measured. In the modified Biel maze, CPEE and control offspring were not different for percent completed trials or time to complete a trial. However, CPEE offspring made more errors (reversals and entering dead ends) in the Biel maze, demonstrating impaired spatial learning and memory. In contrast, CPEE offspring did not have impaired performance of the rewarded-alternation Y-maze task. Therefore, the modified dry-land version of the Biel-maze task, which measures cognitive performance using a complex multi-choice design, is more sensitive in demonstrating CPEE-induced spatial learning and memory deficits compared with a simple, rewarded-alternation Y-maze task.

Background: Ethanol consumption during pregnancy can lead to a range of adverse developmental outcomes in children, termed fetal alcohol spectrum disorder (FASD). Central nervous system injury is a debilitating and widely studied manifestation of chronic prenatal ethanol exposure (CPEE). However, CPEE can also cause structural and functional deficits in metabolic pathways in offspring. Objectives and Methods: This study tested the hypothesis that CPEE increases whole-body adiposity and disrupts pancreatic structure in guinea pig offspring. Pregnant guinea pigs received ethanol (4 g kg−1 maternal body weight per day) or isocaloric-sucrose/pair-feeding (control) for 5 days per week throughout gestation. Results: Male and female CPEE offspring demonstrated growth restriction at birth, followed by a rapid period of catch-up growth before weaning (postnatal day (PD) 1–7). Whole-body magnetic resonance imaging (MRI) in young adult offspring (PD100–140) revealed increased visceral and subcutaneous adiposity produced by CPEE. At the time of killing (PD150–200), CPEE offspring also had increased pancreatic adipocyte area and decreased β-cell insulin-like immunopositive area, suggesting reduced insulin production and/or secretion from pancreatic islets. Conclusion: CPEE causes increased adiposity and pancreatic dysmorphology in offspring, which may signify increased risk for the development of metabolic syndrome and type 2 diabetes mellitus.