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Melling M.A.,Childrens Health Research Institute | Melling M.A.,University of Western Ontario | Friendship C.R.C.,Childrens Health Research Institute | Friendship C.R.C.,University of Western Ontario | And 4 more authors.
Developmental Dynamics | Year: 2013

Background: Retinoic acid signaling is essential for many aspects of early development in vertebrates. To control the levels of signaling, several retinoic acid target genes have been identified that act to suppress retinoic acid signaling in a negative feedback loop. The nuclear protein Ski has been extensively studied for its ability to suppress transforming growth factor-beta (TGF-β) signaling but has also been implicated in the repression of retinoic acid signaling. Results: We demonstrate that ski expression is up-regulated in response to retinoic acid in both early Xenopus embryos and in human cell lines. Blocking retinoic acid signaling using a retinoic acid antagonist results in a corresponding decrease in the levels of ski mRNA. Finally, overexpression of SKI in human cells results in reduced levels of CYP26A1 mRNA, a known target of retinoic acid signaling. Conclusions: Our results, coupled with the known ability of Ski to repress retinoic acid signaling, demonstrate that Ski expression is a novel negative feedback mechanism acting on retinoic acid signaling. Developmental Dynamics 242:604-613, 2013. © 2013 Wiley Periodicals, Inc. Source

Correa R.J.M.,Translational Ovarian Cancer Research Program | Correa R.J.M.,University of Western Ontario | Valdes Y.R.,Translational Ovarian Cancer Research Program | Shepherd T.G.,Translational Ovarian Cancer Research Program | And 3 more authors.
Journal of Ovarian Research | Year: 2015

Abstract Background: Autophagy is a conserved cellular self-digestion mechanism that can either suppress or promote cancer in a context-dependent manner. In ovarian cancer, prevalent mono-allelic deletion of BECN1 (a canonical autophagy-inducer) suggests that autophagy is impaired to promote carcinogenesis and that Beclin-1 is a haploinsufficient tumor suppressor. Nonetheless, autophagy is known to be readily inducible in ovarian cancer cells. We sought to clarify whether Beclin-1 expression is in fact disrupted in ovarian cancer and whether this impacts autophagy regulation. Methods: BECN1 expression levels were assessed using The Cancer Genome Atlas (TCGA) datasets from 398 ovarian high-grade serous cystadenocarcinomas (HGSC) and protein immunoblot data from HGSC samples obtained at our institution. Knockdown of BECN1 and other autophagy-related gene expression was achieved using siRNA in established human ovarian cancer cell lines (CaOV3, OVCAR8, SKOV3, and HeyA8) and a novel early-passage, ascites-derived cell line (iOvCa147-E2). LC3 immunoblot, autophagic flux assays, transmission electron microscopy and fluorescence microscopy were used to assess autophagy. Results: We observed prevalent mono-allelic BECN1 gene deletion (76 %) in TCGA tumors, yet demonstrate for the first time that Beclin-1 protein expression remains relatively unaltered in these and additional samples generated at our institution. Surprisingly, efficient siRNA-mediated Beclin-1 knockdown did not attenuate autophagy induction, whereas knockdown of other autophagy-related genes blocked the process. Beclin-1 knockdown instead decreased cell viability without inducing apoptosis. Conclusions: Taken together, these data demonstrate that despite its sustained expression, Beclin-1 is dispensable for autophagy induction in ovarian tumor cells in vitro, yet may be retained to promote cell viability by a mechanism independent of autophagy or apoptosis regulation. Overall, this work makes novel observations about tumor expression of Beclin-1 and challenges the accepted understanding of its role in regulating autophagy in ovarian cancer. © 2015 Correa et al. Source

Correa R.J.M.,Translational Ovarian Cancer Research Program | Correa R.J.M.,University of Western Ontario | Peart T.,Translational Ovarian Cancer Research Program | Peart T.,University of Western Ontario | And 5 more authors.
Carcinogenesis | Year: 2012

Epithelial ovarian cancer (EOC) metastasis is a direct contributor to high recurrence and low survival for patients with this disease. Metastasis in EOC occurs by cell exfoliation from the primary tumor into the fluid-filled peritoneal cavity, persistence of these cells as non-adherent multicellular aggregates or spheroids and reattachment of spheroids to form secondary lesions. We have recovered native spheroids from ascites fluid and demonstrated that EOC cells within these structures exhibit reduced proliferation, yet regain the capacity to attach and reinitiate cell division. To model this process in vitro for further investigation, primary EOC cells from patient peritoneal fluid were cultured under non-adherent conditions. Here we show that these cells naturally form spheroids resembling those observed in ascites. Spheroids exhibit reduced cell proliferation and a protein expression pattern consistent with cellular quiescence: specifically, decreased phospho-AKT and p45/SKP2 with a concomitant increase in p130/RBL2 and p27 Kip1. However, when spheroids are seeded to an adherent surface, reattachment occurs rapidly and is followed by reinitiation of AKT-dependent cell proliferation. These results were strikingly consistent among numerous clinical specimens and were corroborated in the EOC cell line OVCAR3. Therefore, our data reveal that EOC cells become quiescent when forming spheroids, but reactivate proliferative mechanisms upon attachment to a permissive substratum. Overall, this work utilizes a novel in vitro model of EOC metastasis that employs primary human EOC cells and introduces the important concept of reversible dormancy in EOC pathogenesis. © The Author 2011. Published by Oxford University Press. All rights reserved. Source

Correa R.J.M.,Translational Ovarian Cancer Research Program | Correa R.J.M.,University of Western Ontario | Komar M.,Translational Ovarian Cancer Research Program | Tong J.G.K.,University of Western Ontario | And 7 more authors.
Gynecologic Oncology | Year: 2012

Objective: We propose that metastatic epithelial ovarian cancer (EOC) is a potential therapeutic target for the oncolytic agent, Myxoma virus (MYXV). Methods: Primary EOC cells were isolated from patient ascites and cultured as adherent cells or in suspension using Ultra Low-Attachment dishes. MYXV expressing green fluorescent protein was used to infect cells and spheroids. Infection was monitored by fluorescence microscopy, viral titering and immunoblotting for M-T7 and M130 virus protein expression, and cell viability by alamarBlue assay. Akti-1/2 (5 μM) and rapamycin (20 nM) were used to assay the role of PI3K-AKT signaling in mediating MYXV infection. Results: Ascites-derived EOC cells grown in adherent culture are effectively killed by MYXV infection. EOC cells grown in suspension to form three-dimensional EOC spheroids readily permit MYXV entry into cells, yet are protected from the cytopathic effects of late MYXV infection. Upon reattachment (to model secondary metastasis), EOC spheroids are re-sensitized to MYXV-mediated oncolysis. The critical determinant that facilitates efficient MYXV infection is the presence of an activated PI3K-AKT signaling pathway. Treatment with the specific AKT inhibitor Akti-1/2 reduces infection of monolayer EOC cells and spheroids. Direct infection of freshly-collected ascites demonstrated that 54.5% of patient samples were sensitive to MYXV-mediated oncolytic cell killing. We also demonstrate that factor(s) present in ascites may negatively impact MYXV infection and oncolysis of EOC cells, which may be due to a down-regulation in endogenous AKT activity. Conclusions: Differential activity of AKT serves as the mechanistic basis for regulating MYXV-mediated oncolysis of EOC spheroids during key steps of the metastatic program. In addition, we provide the first evidence that MYXV oncolytic therapy may be efficacious for a significant proportion of ovarian cancer patients with metastatic disease. © 2012 Elsevier Inc. All rights reserved. Source

Peart T.M.,Translational Ovarian Cancer Research Program | Peart T.M.,University of Western Ontario | Correa R.J.M.,Translational Ovarian Cancer Research Program | Correa R.J.M.,University of Western Ontario | And 5 more authors.
Clinical and Experimental Metastasis | Year: 2012

Epithelial ovarian cancer (EOC) cells have the ability to form multi-cellular aggregates in malignant ascites which dramatically alters cell signalling, survival, and metastatic potential. Herein, we demonstrate that patient ascites-derived EOC cells down-regulate endogenous bone morphogenetic protein (BMP) signalling by decreasing BMP ligand expression when grown in suspension culture to form spheroids. Enforced BMP signalling in these cells via constitutively-active BMP type I ALK3 QD receptor expression causes the formation of smaller, more loosely-aggregated spheroids. Additionally, ALK3 QD-expressing spheroids have an increased rate of adhesion and dispersion upon reattachment to substratum. Inhibition of endogenous BMP signalling using recombinant Noggin or small molecule inhibitor LDN-193189, on the other hand, opposed these phenotypic changes. To identify potential targets that impact the phenotype of EOC spheroids due to activated BMP signalling, we performed genome-wide expression analyses using Affymetrix arrays. Using the online Connectivity Map resource, the BMP signalling gene expression signature revealed that the AKT pathway is induced by activated BMP signalling in EOC cells; this finding was further validated by phospho-AKT immuno-blotting. In fact, treatment of EOC spheroids with an AKT inhibitor, Akti-1/2, reduced BMP-stimulated cell dispersion during reattachment as compared to controls. Thus, we have identified AKT as being one important downstream component of activated BMP signalling on EOC spheroid pathobiology, which may have important implications on the metastatic potential of this malignancy. © 2012 Springer Science+Business Media B.V. Source

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