Publications by authors named "Rozanne Arulanandam"

37 Publications

Modulation of Akt vs Stat3 activity by the focal adhesion kinase in non-neoplastic mouse fibroblasts.

Exp Cell Res 2021 Jul 3;404(1):112601. Epub 2021 May 3.

Department of Biomedical and Molecular Sciences and Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, K7L 3N6, Canada.

Adhesion of cells to each other and to the extracellular matrix (ECM) are both required for cellular functions. Cell-to-cell adhesion is mediated by cadherins and their engagement triggers the activation of Stat3, which offers a potent survival signal. Adhesion to the ECM on the other hand, activates FAK which attracts and activates Src, as well as receptor tyrosine kinases (RTKs), the PI3k/Akt and Ras/Erk pathways. However, the effect of cell density upon FAK and Akt activity has not been examined. We now demonstrate that, interestingly, despite being potent Stat3 activators, Src and RTKs are unable to activate Stat3 in sparsely growing (i.e., without cadherin engagement), non-neoplastic cells attached to the ECM. In contrast, cell aggregation (i.e., cadherin engagement in the absence of adhesion to a solid substratum) was found to activate both Stat3 and Akt. Pharmacologic or genetic reduction of FAK activity abolished Akt activity at low densities, indicating that FAK is an important activator of Akt in this setting. Notably, FAK knockout increased cellular sensitivity to the Stat3 inhibitor CPA7, while FAK reintroduction restored resistance to this drug. These findings suggest a complementary role of integrin/FAK/Akt and cadherin/Stat3-mediated pro-survival pathways, which may be of significance during neoplastic transformation and metastasis.
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http://dx.doi.org/10.1016/j.yexcr.2021.112601DOI Listing
July 2021

SARS-CoV-2 S1 NanoBiT: A nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition.

Biosens Bioelectron 2021 May 2;180:113122. Epub 2021 Mar 2.

Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, K1H 8M5, Canada. Electronic address:

As the COVID-19 pandemic continues, there is an imminent need for rapid diagnostic tools and effective antivirals targeting SARS-CoV-2. We have developed a novel bioluminescence-based biosensor to probe a key host-virus interaction during viral entry: the binding of SARS-CoV-2 viral spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2). Derived from Nanoluciferase binary technology (NanoBiT), the biosensor is composed of Nanoluciferase split into two complementary subunits, Large BiT and Small BiT, fused to the Spike S1 domain of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. The ACE2-S1 interaction results in reassembly of functional Nanoluciferase, which catalyzes a bioluminescent reaction that can be assayed in a highly sensitive and specific manner. We demonstrate the biosensor's large dynamic range, enhanced thermostability and pH tolerance. In addition, we show the biosensor's versatility towards the high-throughput screening of drugs which disrupt the ACE2-S1 interaction, as well as its ability to act as a surrogate virus neutralization assay. Results obtained with our biosensor correlate well with those obtained with a Spike-pseudotyped lentivirus assay. This rapid in vitro tool does not require infectious virus and should enable the timely development of antiviral modalities targeting SARS-CoV-2 entry.
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http://dx.doi.org/10.1016/j.bios.2021.113122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921772PMC
May 2021

Characterization of Critical Determinants of ACE2-SARS CoV-2 RBD Interaction.

Int J Mol Sci 2021 Feb 25;22(5). Epub 2021 Feb 25.

Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. In this study, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2, by utilizing our recently developed NanoBiT technology-based biosensor as well as pseudotyped-virus infectivity assays. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, efficacy of competitive inhibitors, as well as neutralizing antibody activity. We also look at the implications the mutations may have on virus transmissibility, host susceptibility, and the virus transmission path to humans. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.
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http://dx.doi.org/10.3390/ijms22052268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956771PMC
February 2021

Combining vanadyl sulfate with Newcastle disease virus potentiates rapid innate immune-mediated regression with curative potential in murine cancer models.

Mol Ther Oncolytics 2021 Mar 21;20:306-324. Epub 2021 Jan 21.

Department of Pathobiology, University of Guelph, Guelph, ON N1G 2WI, Canada.

The avian paramyxovirus, Newcastle disease virus (NDV), is a promising oncolytic agent that has been shown to be safe and effective in a variety of pre-clinical cancer models and human clinical trials. NDV preferentially replicates in tumor cells due to signaling defects in apoptotic and antiviral pathways acquired during the transformation process and is a potent immunostimulatory agent. However, when used as a monotherapy NDV lacks the ability to consistently generate durable remissions. Here we investigate the use of viral sensitizer-mediated combination therapy to enhance the anti-neoplastic efficacy of NDV. Intratumoral injection of vanadyl sulfate, a pan-inhibitor of protein tyrosine phosphatases, in combination with NDV significantly increased the number and activation status of natural killer (NK) cells in the tumor microenvironment, concomitant with increased expression of interferon-β, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1, leading to rapid tumor regression and long-term cures in mice bearing syngeneic B16-F10 melanomas. The anti-tumor efficacy of this combination therapy was abrogated when NK cells were depleted and when interferon-β expression was transiently suppressed. Tumor-specific CD8 T cell responses were not detected, nor were mice whose tumors regressed protected from re-challenge. This suggested efficacy of the combination therapy predominantly relied on the innate immune system. Importantly, efficacy was not limited to melanoma; it was also demonstrated in a murine prostate cancer model. Taken together, these results suggest that combining NDV with vanadyl sulfate potentiates an innate immune response that can potentiate rapid clearance of tumors, with type I interferon signaling and NK cells being important mechanisms of action.
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http://dx.doi.org/10.1016/j.omto.2021.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868934PMC
March 2021

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

Mol Ther 2021 06 10;29(6):1984-2000. Epub 2021 Feb 10.

Center for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON K1H 8M5, Canada. Electronic address:

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.
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http://dx.doi.org/10.1016/j.ymthe.2021.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872859PMC
June 2021

The strategic combination of trastuzumab emtansine with oncolytic rhabdoviruses leads to therapeutic synergy.

Commun Biol 2020 05 22;3(1):254. Epub 2020 May 22.

Centre for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada.

We have demonstrated that microtubule destabilizing agents (MDAs) can sensitize tumors to oncolytic vesicular stomatitis virus (VSVΔ51) in various preclinical models of cancer. The clinically approved T-DM1 (Kadcyla®) is an antibody-drug conjugate consisting of HER2-targeting trastuzumab linked to the potent MDA and maytansine derivative DM1. We reveal that combining T-DM1 with VSVΔ51 leads to increased viral spread and tumor killing in trastuzumab-binding, VSVΔ51-resistant cancer cells. In vivo, co-treatment of VSVΔ51 and T-DM1 increased overall survival in HER2-overexpressing, but trastuzumab-refractory, JIMT1 human breast cancer xenografts compared to monotherapies. Furthermore, viral spread in cultured HER2 human ovarian cancer patient-derived ascites samples was enhanced by the combination of VSVΔ51 and T-DM1. Our data using the clinically approved Kadcyla® in combination with VSVΔ51 demonstrates proof of concept that targeted delivery of a viral-sensitizing molecule using an antibody-drug conjugate can enhance oncolytic virus activity and provides rationale for translation of this approach.
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http://dx.doi.org/10.1038/s42003-020-0972-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244474PMC
May 2020

Enhancement of oncolytic virotherapy by vanadium(V) dipicolinates.

Biometals 2019 06 17;32(3):545-561. Epub 2019 Jun 17.

Department of Chemistry, Colorado State University, Fort Collins, CO, USA.

Oncolytic viruses rewire the immune system and can lead to long-lasting antitumor defenses against primary and metastatic tumors. However, results from clinical studies have shown heterogeneity in responses suggesting that multiplexed approaches may be necessary to consistently generate positive outcomes in patients. To this end, we explored the combination of oncolytic rhabdovirus VSV∆51 with vanadium(V) dipicolinate derivatives, which have already been explored for their antidiabetic properties in animal models. The combination of vanadium-based dipicolinate compounds with VSV∆51 significantly increased viral replication and cytotoxicity in the human renal cell carcinoma cell line 786-0. The effects of three vanadium(V)-dipicolinate coordination complexes ([VOdipic], [VOdipic-OH] and [VOdipic-Cl] with -OH or -Cl in the para position) were compared to that of the simple salts using spectroscopy and speciation profiles. Like the vanadate salts and the vanadyl cation, all dioxovanadium(V) dipicolinate complexes tested were found to increase viral infection and cytotoxicity when used in combination with VSV∆51. Viral sensitization is dependent on the vanadium since free dipicolinate ligands exerted no effect on viral infection and viability. The ability of these complexes to interact with interfaces and the stability of the complexes were evaluated under physiological conditions. Results indicate that these complexes undergo hydrolysis in cell culture media thereby generating vanadate. The vanadium dipicolinate derivatives in the context of immunovirotherapy shares similarities with previous studies exploring the antidiabetic properties of the compounds. The synergy between vanadium compounds and the oncolytic virus suggests that these compounds may be valuable in the development of novel and effective pharmaco-viral therapies.
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http://dx.doi.org/10.1007/s10534-019-00200-9DOI Listing
June 2019

Regulation of Differentiation of HC11 Mouse Breast Epithelial Cells by the Signal Transducer and Activator of Transcription-3.

Anticancer Res 2019 Jun;39(6):2749-2756

Department of Biomedical and Molecular Sciences and Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada.

Background/aim: The differentiation of the mouse breast epithelial cell line HC11 is known to require confluence as well as the addition of hydrocortisone, insulin and prolactin.

Materials And Methods: Since confluence, which triggers the engagement of the cell-to-cell adhesion molecule E-cadherin, induces a dramatic increase in the activity of signal transducer and activator of transcription-3 (Stat3), we examined the role of Stat3 in HC11 cell differentiation.

Results: Stat3 inhibition abolished differentiation, indicating that Stat3 activity is critically required. However, expression of the mutationally activated form of Stat3 (Stat3C), rather than promoting, it was found to block cell differentiation, even when expressed in low levels, and in the absence of full neoplastic conversion.

Conclusion: The strength of the E-cadherin/Stat3 signal is key for the outcome of the differentiation process.
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http://dx.doi.org/10.21873/anticanres.13401DOI Listing
June 2019

Effect of caveolin-1 on Stat3-ptyr705 levels in breast and lung carcinoma cells.

Biochem Cell Biol 2019 10 15;97(5):638-646. Epub 2019 Apr 15.

Department of Biomedical and Molecular Sciences, Pathology and Molecular Medicine, and Queen's University Cancer Research Institute, Queen's University, Kingston, ON K7L 3N6, Canada.

We recently demonstrated that Cav1 (caveolin-1) is a negative regulator of Stat3 (signal transducer and activator of transcription-3) activity in mouse fibroblasts and human lung carcinoma SHP77 cells. We now examined whether the cellular context may affect their levels as well as the relationship between them, by assessing Cav1 and Stat3-ptyr705 amounts in different cell lines. In MDA-MB-231, A549, and HaCat cells, Cav1 levels were high and Stat3-ptyr705 levels were low, consistent with the notion of a negative effect of endogenous Cav1 on Stat3-ptyr705 levels in these lines. In addition, manipulation of Cav1 levels revealed a negative effect in MCF7 and mouse fibroblast cells, while Cav1 upregulation induced apoptosis in MCF7 cells. In contrast, however, line MRC9 had high Cav1 and high Stat3-ptyr705 levels, indicating that high Cav1 is insufficient to reduce Stat3-ptyr705 levels in this line. MCF7 and LuCi6 cells had very low Cav1 and Stat3-ptyr705 levels, indicating that the low Stat3-ptyr705 can be independent from Cav1 levels altogether. Our results reveal a further level of complexity in the relationship between Cav1 and Stat3-ptyr705 than previously thought. In addition, we demonstrate that in a feedback loop, Stat3 inhibition upregulates Cav1 in HeLa cells but not in other lines tested.
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http://dx.doi.org/10.1139/bcb-2018-0367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038897PMC
October 2019

Regulation of HC11 mouse breast epithelial cell differentiation by the E-cadherin/Rac axis.

Exp Cell Res 2017 12 13;361(1):112-125. Epub 2017 Oct 13.

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6; Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada K7L3N6. Electronic address:

It was previously demonstrated that differentiation of some established breast epithelial cell lines requires confluence and stimulation with hydrocortisone, insulin and prolactin inducers. We and others previously demonstrated that E-cadherin engagement, which is favored under conditions of confluence, increases the levels and activity of the Rac small GTPase. To investigate the functional relationship between the transforming ability of Rac and its role as an integral component of the differentiative E-cadherin signaling pathway, we introduced a mutationally activated form of Rac, Rac, into the mouse breast epithelium-derived cell line, HC11. Our results demonstrate that the strength of the Rac signal is key for the outcome of the differentiation process; cRac1 is critically required for differentiation, and at low levels, mutationally activated Rac is able to increase differentiation, presumably reinforcing the E-cadherin/Rac differentiative signal. However, high Rac expression blocked differentiation concomitant with E-cadherin downregulation, while inducing neoplastic transformation. Therefore, the intensity of the Rac signal is a central determinant in the balance between cell proliferation vs differentiation, two fundamentally opposed processes, a finding which could also have important therapeutic implications.
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http://dx.doi.org/10.1016/j.yexcr.2017.10.008DOI Listing
December 2017

Oncolytic Maraba Virus MG1 as a Treatment for Sarcoma.

Int J Cancer 2017 09 21;141(6):1257-1264. Epub 2017 Jun 21.

Centre for Innovative Cancer research, Ottawa Hospital Research Institute, Ottawa, ON, KlH 8L6, Canada.

The poor prognosis of patients with advanced bone and soft-tissue sarcoma has not changed in the past several decades, highlighting the necessity for new therapeutic approaches. Immunotherapies, including oncolytic viral (OV) therapy, have shown great promise in a number of clinical trials for a variety of tumor types. However, the effective application of OV in treating sarcoma still remains to be demonstrated. Although few pre-clinical studies using distinct OVs have been performed and demonstrated therapeutic benefit in sarcoma models, a side-by-side comparison of clinically relevant OV platforms has not been performed. Four clinically relevant OV platforms (Reovirus, Vaccinia virus, Herpes-simplex virus and Rhabdovirus) were screened for their ability to infect and kill human and canine sarcoma cell lines in vitro, and human sarcoma specimens ex vivo. In vivo treatment efficacy was tested in a murine model. The rhabdovirus MG1 demonstrated the highest potency in vitro. Ex vivo, MG1 productively infected more than 80% of human sarcoma tissues tested, and treatment in vivo led to a significant increase in long-lasting cures in sarcoma-bearing mice. Importantly, MG1 treatment induced the generation of memory immune response that provided protection against a subsequent tumor challenge. This study opens the door for the use of MG1-based oncolytic immunotherapy strategies as treatment for sarcoma or as a component of a combined therapy.
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http://dx.doi.org/10.1002/ijc.30813DOI Listing
September 2017

Enhancing Expression of Functional Human Sodium Iodide Symporter and Somatostatin Receptor in Recombinant Oncolytic Vaccinia Virus for In Vivo Imaging of Tumors.

J Nucl Med 2017 02 15;58(2):221-227. Epub 2016 Sep 15.

Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada

Oncolytic virus (OV) therapy has emerged as a novel tool in our therapeutic arsenals for fighting cancer. As a live biologic agent, OV has the ability to target and selectively amplify at the tumor sites. We have reported that a vaccinia-based OV (Pexa-Vec) has shown good efficacy in preclinical models and in clinical trials. To give an additional tool to clinicians to allow both treatment of the tumor and improved visualization of tumor margins, we developed new viral-based platforms with 2 specific gene reporters.

Methods: We incorporated the human sodium iodide symporter (hNIS) and the human somatostatin receptor 2 (hSSR2) in the vaccinia-based OV and tested viral constructs for their abilities to track and treat tumor development in vivo.

Results: Early and high-level expression of hNIS is detrimental to the recombinant virus, leading to the aggregation of hNIS protein and early cell death. Putting hNIS under a late synthetic promoter allowed a higher functional expression of the protein and much stronger I or Tc uptake. In vivo, the hNIS-containing virus infected and amplified in the tumor site, showing a better efficacy than the parental virus. The hNIS expression at the tumor site allowed for the imaging of viral infection and tumor regression. Similarly, hSSR2-containing OV vaccinia infected and lysed cancer cells.

Conclusion: When tumor-bearing mice were given hNIS- and hSSR2-containing OV, Tc and In signals coalesced at the tumor, highlighting the power of using these viruses for tumor diagnosis and treatment.
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http://dx.doi.org/10.2967/jnumed.116.180463DOI Listing
February 2017

Combination of Paclitaxel and MG1 oncolytic virus as a successful strategy for breast cancer treatment.

Breast Cancer Res 2016 08 8;18(1):83. Epub 2016 Aug 8.

Ottawa Hospital Research Institute, Centre for Innovative Cancer Research, Ottawa, K1H 8L6, Canada.

Background: Breast cancer is the most common malignant disease amongst Western women. The lack of treatment options for patients with chemotherapy-resistant or recurrent cancers is pushing the field toward the rapid development of novel therapies. The use of oncolytic viruses is a promising approach for the treatment of disseminated diseases like breast cancer, with the first candidate recently approved by the Food and Drug Administration for use in patients. In this report, we demonstrate the compatibility of oncolytic virotherapy and chemotherapy using various murine breast cancer models. This one-two punch has been explored in the past by several groups with different viruses and drugs and was shown to be a successful approach. Our strategy is to combine Paclitaxel, one of the most common drugs used to treat patients with breast cancer, and the oncolytic Rhabdovirus Maraba-MG1, a clinical trial candidate in a study currently recruiting patients with late-stage metastatic cancer.

Methods: We used the EMT6, 4 T1 and E0771 murine breast cancer models to evaluate in vitro and in vivo the effects of co-treatment with MG1 and Paclitaxel. Treatment-induced cytotoxicity was assessed and plaque assays, flow cytometry, microscopy and immunocytochemistry analysis were performed to quantify virus production and transgene expression. Orthotopically implanted tumors were measured during and after treatment to evaluate efficacy and Kaplan-Meier survival curves were generated.

Results: Our data demonstrate not only the compatibility of the treatments, but also their synergistic cytopathic activity. With Paclitaxel, EMT6 and 4 T1 tumors demonstrated increased virus production both in vitro and in vivo. Our results also show that Paclitaxel does not impair the safety profile of the virus treatment. Importantly, when combined, MG1 and the drug controlled tumor growth and prolonged survival.

Conclusions: The combination of MG1 and Paclitaxel improved efficacy in all of the breast cancer models we tested and thus is a promising alternative approach for the treatment of patients with refractory breast cancer. Our strategy has potential for rapid translation to the clinic, given the current clinical status of both agents.
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http://dx.doi.org/10.1186/s13058-016-0744-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4977613PMC
August 2016

Cell-cell and cell-matrix adhesion in survival and metastasis: Stat3 versus Akt.

Biomol Concepts 2015 Dec;6(5-6):383-99

Both cell-cell and cell-matrix adhesion are important for epithelial cell differentiation and function. Classical cadherins mediate cell to cell interactions and are potent activators of the signal transducer and activator of transcription (Stat3), thereby offering survival signaling. While the epithelial (E)-cadherin is required for cells to remain tightly associated within differentiated epithelial tissues, cadherin-11 promotes invasion and metastasis, preferentially to the bone. Cell adhesion to the extracellular matrix is mediated through the integrin receptors that bind to the focal adhesion kinase (FAK)/Src complex, thus activating downstream effectors such as Ras/Erk1/2 and PI3k/Akt, but not Stat3. Therefore, at high densities of cultured cells or in epithelial tissues, co-ordinate activation of the complementary cadherin/Stat3 and integrin/FAK pathways can greatly enhance survival and growth of tumor cells. In neoplastically transformed cells on the other hand, a variety of oncogenes including activated Src or receptor tyrosine kinases, activate both pathways. Still, most single-agent therapies directed against these signaling pathways have proven disappointing in the clinic. Combined targeting of the Src/FAK and Stat3 pathways with inhibitory drugs would be expected to have greater efficacy in inhibiting tumor cell survival, and enhancing sensitivity to conventional cytotoxic drugs for treatment of metastatic disease.
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http://dx.doi.org/10.1515/bmc-2015-0022DOI Listing
December 2015

VEGF-Mediated Induction of PRD1-BF1/Blimp1 Expression Sensitizes Tumor Vasculature to Oncolytic Virus Infection.

Cancer Cell 2015 Aug 23;28(2):210-24. Epub 2015 Jul 23.

Centre for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada. Electronic address:

Oncolytic viruses designed to attack malignant cells can in addition infect and destroy tumor vascular endothelial cells. We show here that this expanded tropism of oncolytic vaccinia virus to the endothelial compartment is a consequence of VEGF-mediated suppression of the intrinsic antiviral response. VEGF/VEGFR2 signaling through Erk1/2 and Stat3 leads to upregulation, nuclear localization, and activation of the transcription repressor PRD1-BF1/Blimp1. PRD1-BF1 does not contribute to the mitogenic effects of VEGF, but directly represses genes involved in type I interferon (IFN)-mediated antiviral signaling. In vivo suppression of VEGF signaling diminishes PRD1-BF1/Blimp1 expression in tumor vasculature and inhibits intravenously administered oncolytic vaccinia delivery to and consequent spread within the tumor.
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http://dx.doi.org/10.1016/j.ccell.2015.06.009DOI Listing
August 2015

Reciprocal cellular cross-talk within the tumor microenvironment promotes oncolytic virus activity.

Nat Med 2015 May 20;21(5):530-6. Epub 2015 Apr 20.

1] Centre for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. [2] Department of Biochemistry, Immunology and Microbiology, University of Ottawa, Ottawa, Ontario, Canada.

Tumors are complex ecosystems composed of networks of interacting 'normal' and malignant cells. It is well recognized that cytokine-mediated cross-talk between normal stromal cells, including cancer-associated fibroblasts (CAFs), vascular endothelial cells, immune cells, and cancer cells, influences all aspects of tumor biology. Here we demonstrate that the cross-talk between CAFs and cancer cells leads to enhanced growth of oncolytic virus (OV)-based therapeutics. Transforming growth factor-β (TGF-β) produced by tumor cells reprogrammed CAFs, dampened their steady-state level of antiviral transcripts and rendered them sensitive to virus infection. In turn, CAFs produced high levels of fibroblast growth factor 2 (FGF2), initiating a signaling cascade in cancer cells that reduced retinoic acid-inducible gene I (RIG-I) expression and impeded the ability of malignant cells to detect and respond to virus. In xenografts derived from individuals with pancreatic cancer, the expression of FGF2 correlated with the susceptibility of the cancer cells to OV infection, and local application of FGF2 to resistant tumor samples sensitized them to virotherapy both in vitro and in vivo. An OV engineered to express FGF2 was safe in tumor-bearing mice, showed improved therapeutic efficacy compared to parental virus and merits consideration for clinical testing.
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http://dx.doi.org/10.1038/nm.3848DOI Listing
May 2015

Microtubule disruption synergizes with oncolytic virotherapy by inhibiting interferon translation and potentiating bystander killing.

Nat Commun 2015 Mar 30;6:6410. Epub 2015 Mar 30.

Center for Innovative Cancer Research, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6.

In this study, we show that several microtubule-destabilizing agents used for decades for treatment of cancer and other diseases also sensitize cancer cells to oncolytic rhabdoviruses and improve therapeutic outcomes in resistant murine cancer models. Drug-induced microtubule destabilization leads to superior viral spread in cancer cells by disrupting type I IFN mRNA translation, leading to decreased IFN protein expression and secretion. Furthermore, microtubule-destabilizing agents specifically promote cancer cell death following stimulation by a subset of infection-induced cytokines, thereby increasing viral bystander effects. This study reveals a previously unappreciated role for microtubule structures in the regulation of the innate cellular antiviral response and demonstrates that unexpected combinations of approved chemotherapeutics and biological agents can lead to improved therapeutic outcomes.
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http://dx.doi.org/10.1038/ncomms7410DOI Listing
March 2015

Stat3 and gap junctions in normal and lung cancer cells.

Cancers (Basel) 2014 Mar 25;6(2):646-62. Epub 2014 Mar 25.

Department of Pathology, Queen's University, Kingston, ON K7L 3N6, Canada.

Gap junctions are channels linking the interiors of neighboring cells. A reduction in gap junctional intercellular communication (GJIC) correlates with high cell proliferation, while oncogene products such as Src suppress GJIC, through the Ras/Raf/Erk and other effector pathways. High Src activity was found to correlate with high levels of the Src effector, Signal Transducer and Activator of Transcription-3 (Stat3) in its tyrosine-705 phosphorylated, i.e., transcriptionally activated form, in the majority of Non-Small Cell Lung Cancer lines examined. However, Stat3 inhibition did not restore GJIC in lines with high Src activity. In the contrary, Stat3 inhibition in normal cells or in lines with low Src activity and high GJIC eliminated gap junctional communication. Therefore, despite the fact that Stat3 is growth promoting and in an activated form acts like an oncogene, it is actually required for junctional permeability.
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http://dx.doi.org/10.3390/cancers6020646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4074796PMC
March 2014

Engaged for survival: From cadherin ligation to STAT3 activation.

JAKSTAT 2013 Oct 6;2(4):e27363. Epub 2013 Dec 6.

Department of Pathology; Queen's University; Kingston, ON Canada ; Department of Biomedical and Molecular Sciences; Queen's University; Kingston, ON Canada.

In normal tissues or tumors, cells have extensive opportunities for adhesion to their neighbors. This state is mimicked by dense cell cultures. In this review, we integrate some recent findings on a key signal transducer, STAT3 (signal transducer and activator of transcription-3), whose activity is dramatically increased following cadherin-mediated cell to cell adhesion. Cadherin engagement, favored in dense cell cultures, causes a dramatic increase in total Rac/Cdc42 protein levels through inhibition of proteasomal degradation, which is followed by activation of IL-6 and STAT3. The cadherin/Rac/IL-6/STAT3 axis offers a potent survival signal that is a prerequisite for neoplastic transformation, as well as normal tissue function.
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http://dx.doi.org/10.4161/jkst.27363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891631PMC
October 2013

Panorama from the oncolytic virotherapy summit.

Mol Ther 2013 Oct;21(10):1814-8

McMaster Immunology Research Center, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.

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http://dx.doi.org/10.1038/mt.2013.207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808141PMC
October 2013

Tumor vascularization is critical for oncolytic vaccinia virus treatment of peritoneal carcinomatosis.

Int J Cancer 2014 Feb 29;134(3):717-30. Epub 2013 Aug 29.

Division of Experimental Therapeutics, Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada; Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.

Peritoneal carcinomatosis (PC) represents a significant clinical challenge for which there are few treatment options. Oncolytic viruses are ideal candidates for PC treatment because of their high tumor specificity, excellent safety profile and suitability for peritoneal delivery. Here, we described the use of vvDD-SR-RFP, a recombinant vaccinia virus, in xenograft and syngeneic models of colorectal PC. Colorectal cancer cell lines were highly susceptible to vvDD-SR-RFP replication and cytotoxicity. Intraperitoneal delivery of vvDD-SR-RFP on Day 12 to mice with colorectal carcinomatosis significantly improved survival whereas survival was not improved following virus treatment on Day 8, when tumors were smaller. Immunohistochemistry revealed early tumors had a poorly distributed network of blood vessels and lower proliferation index compared to later tumors. Virus infection was also restricted to tumor rims following Day 8 treatment, whereas it was disseminated in tumors treated on Day 12. Additionally, direct infection of tumor endothelium was observed and virus infection correlated with a loss of endothelial staining and induction of cell death. Our results demonstrate that tumor vasculature has a critical role in virus delivery and tumor response. This will have significant implications in the clinical setting, both in understanding timing of therapies and in designing combination treatment strategies.
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http://dx.doi.org/10.1002/ijc.28395DOI Listing
February 2014

Oncolytic vaccinia virus disrupts tumor-associated vasculature in humans.

Cancer Res 2013 Feb 7;73(4):1265-75. Epub 2013 Feb 7.

Jennerex Inc., 450 Sansome, San Francisco, CA 94111, USA.

Efforts to selectively target and disrupt established tumor vasculature have largely failed to date. We hypothesized that a vaccinia virus engineered to target cells with activation of the ras/MAPK signaling pathway (JX-594) could specifically infect and express transgenes (hGM-CSF, β-galactosidase) in tumor-associated vascular endothelial cells in humans. Efficient replication and transgene expression in normal human endothelial cells in vitro required either VEGF or FGF-2 stimulation. Intravenous infusion in mice resulted in virus replication in tumor-associated endothelial cells, disruption of tumor blood flow, and hypoxia within 48 hours; massive tumor necrosis ensued within 5 days. Normal vessels were not affected. In patients treated with intravenous JX-594 in a phase I clinical trial, we showed dose-dependent endothelial cell infection and transgene expression in tumor biopsies of diverse histologies. Finally, patients with advanced hepatocellular carcinoma, a hypervascular and VEGF-rich tumor type, were treated with JX-594 on phase II clinical trials. JX-594 treatment caused disruption of tumor perfusion as early as 5 days in both VEGF receptor inhibitor-naïve and -refractory patients. Toxicities to normal blood vessels or to wound healing were not evident clinically or on MRI scans. This platform technology opens up the possibility of multifunctional engineered vaccinia products that selectively target and infect tumor-associated endothelial cells, as well as cancer cells, resulting in transgene expression, vasculature disruption, and tumor destruction in humans systemically.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-2687DOI Listing
February 2013

Stat3 is a positive regulator of gap junctional intercellular communication in cultured, human lung carcinoma cells.

BMC Cancer 2012 Dec 18;12:605. Epub 2012 Dec 18.

Department of Microbiology, Queen's University, Kingston, Ontario, K7L3N6, Canada.

Background: Neoplastic transformation of cultured cells by a number of oncogenes such as src suppresses gap junctional, intercellular communication (GJIC); however, the role of Src and its effector Signal transducer and activator of transcription-3 (Stat3) upon GJIC in non small cell lung cancer (NSCLC) has not been defined. Immunohistochemical analysis revealed high Src activity in NSCLC biopsy samples compared to normal tissues. Here we explored the potential effect of Src and Stat3 upon GJIC, by assessing the levels of tyr418-phosphorylated Src and tyr705-phosphorylated Stat3, respectively, in a panel of NSCLC cell lines.

Methods: Gap junctional communication was examined by electroporating the fluorescent dye Lucifer yellow into cells grown on a transparent electrode, followed by observation of the migration of the dye to the adjacent, non-electroporated cells under fluorescence illumination.

Results: An inverse relationship between Src activity levels and GJIC was noted; in five lines with high Src activity GJIC was absent, while two lines with extensive GJIC (QU-DB and SK-LuCi6) had low Src levels, similar to a non-transformed, immortalised lung epithelial cell line. Interestingly, examination of the mechanism indicated that Stat3 inhibition in any of the NSCLC lines expressing high endogenous Src activity levels, or in cells where Src was exogenously transduced, did not restore GJIC. On the contrary, Stat3 downregulation in immortalised lung epithelial cells or in the NSCLC lines displaying extensive GJIC actually suppressed junctional permeability.

Conclusions: Our findings demonstrate that although Stat3 is generally growth promoting and in an activated form it can act as an oncogene, it is actually required for gap junctional communication both in nontransformed lung epithelial cells and in certain lung cancer lines that retain extensive GJIC.
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http://dx.doi.org/10.1186/1471-2407-12-605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575370PMC
December 2012

Harnessing oncolytic virus-mediated antitumor immunity in an infected cell vaccine.

Mol Ther 2012 Sep 3;20(9):1791-9. Epub 2012 Jul 3.

Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.

Treatment of permissive tumors with the oncolytic virus (OV) VSV-Δ51 leads to a robust antitumor T-cell response, which contributes to efficacy; however, many tumors are not permissive to in vivo treatment with VSV-Δ51. In an attempt to channel the immune stimulatory properties of VSV-Δ51 and broaden the scope of tumors that can be treated by an OV, we have developed a potent oncolytic vaccine platform, consisting of tumor cells infected with VSV-Δ51. We demonstrate that prophylactic immunization with this infected cell vaccine (ICV) protected mice from subsequent tumor challenge, and expression of granulocyte-monocyte colony stimulating factor (GM-CSF) by the virus (VSVgm-ICV) increased efficacy. Immunization with VSVgm-ICV in the VSV-resistant B16-F10 model induced maturation of dendritic and natural killer (NK) cell populations. The challenge tumor is rapidly infiltrated by a large number of interferon γ (IFNγ)-producing T and NK cells. Finally, we demonstrate that this approach is robust enough to control the growth of established tumors. This strategy is broadly applicable because of VSV's extremely broad tropism, allowing nearly all cell types to be infected at high multiplicities of infection in vitro, where the virus replication kinetics outpace the cellular IFN response. It is also personalized to the unique tumor antigen(s) displayed by the cancer cell.
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http://dx.doi.org/10.1038/mt.2012.128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437573PMC
September 2012

The R(h)oads to Stat3: Stat3 activation by the Rho GTPases.

Exp Cell Res 2011 Aug 18;317(13):1787-95. Epub 2011 May 18.

Department of Microbiology and Immunology and Pathology, Queen's University, Kingston, Ontario, Canada.

The signal transducer and activator of transcription-3 (Stat3) is a member of the STAT family of cytoplasmic transcription factors. Overactivation of Stat3 is detected with high frequency in human cancer and is considered a molecular abnormality that supports the tumor phenotype. Despite concerted investigative efforts, the molecular mechanisms leading to the aberrant Stat3 activation and Stat3-mediated transformation and tumorigenesis are still not clearly defined. Recent evidence reveals a crosstalk close relationship between Stat3 signaling and members of the Rho family of small GTPases, including Rac1, Cdc42 and RhoA. Specifically, Rac1, acting in a complex with the MgcRacGAP (male germ cell RacGAP), promotes tyrosine phosphorylation of Stat3 by the IL6-receptor family/Jak kinase complex, as well as its translocation to the nucleus. Studies have further revealed that the mutational activation of Rac1 and Cdc42 results in Stat3 activation, which occurs in part through the upregulation of IL6 family cytokines that in turn stimulates Stat3 through the Jak kinases. Interestingly, evidence also shows that the engagement of cadherins, cell to cell adhesion molecules, specifically induces a striking increase in Rac1 and Cdc42 protein levels and activity, which in turn results in Stat3 activation. In this review we integrate recent findings clarifying the role of the Rho family GTPases in Stat3 activation in the context of malignant progression.
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http://dx.doi.org/10.1016/j.yexcr.2011.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3129747PMC
August 2011

Housekeeping genes; expression levels may change with density of cultured cells.

J Immunol Methods 2010 Apr 19;355(1-2):76-9. Epub 2010 Feb 19.

Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6.

Western blotting is a powerful technique to characterize a multitude of cellular proteins. As an internal control, the blots are commonly probed for "housekeeping" gene products. In this communication, we show that cell confluence significantly affects the levels of two such widely used proteins, alpha-tubulin and Glyceraldehyde-3-Phosphate Dehydrogenase. On the other hand the levels of heat-shock protein-90 and beta-actin remained unchanged at a wide range of cell densities, making these proteins into more reliable loading controls.
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http://dx.doi.org/10.1016/j.jim.2010.02.006DOI Listing
April 2010

The simian virus 40 large tumor antigen activates cSrc and requires cSrc for full neoplastic transformation.

Anticancer Res 2010 Jan;30(1):47-53

Department of Microbiology and Immunology, Queen's University, Botterell Hall, Rm. 713, Kingston, Ontario K7L3N6, Canada.

Aim: To investigate the role of the cellular protooncogene product, cSrc, in neoplastic transformation by the large tumor antigen of simian virus 40 (TAg), the ability of TAg to increase cSrc activity was examined.

Materials And Methods: cSrc activity was measured in cells expressing wild-type or mutant TAg and compared to the parental line.

Results: The results indicated that TAg expression in mouse 3T3 fibroblasts causes a dramatic increase in cSrc activity, a finding which establishes TAg as a cSrc activator. This ability depended upon a TAg, intact retinoblastoma-susceptibility gene product (Rb) family-binding site. In addition, genetic ablation of pRb in mouse fibroblasts increased cSrc activity, suggesting that pRb inactivation by TAg might be responsible for the observed cSrc activation. Furthermore, down-regulation or genetic ablation of cSrc alone, or together with the Src family members, Yes and Fyn, caused a dramatic reduction in the ability of TAg to transform mouse fibroblasts.

Conclusion: Taken together, these findings suggest for the first time that cSrc is part of an important pathway emanating from TAg and leading to neoplastic conversion.
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January 2010

Beyond structure, to survival: activation of Stat3 by cadherin engagement.

Biochem Cell Biol 2009 Dec;87(6):835-43

Department of Microbiology and Immunology, and Cancer Research Institute, Queen's University, Kingston, ON K7L 3N6.

Cells in normal tissues or in tumors have extensive opportunities for adhesion to their neighbors and the importance of cell to cell contact in the study of fundamental cellular processes is beginning to emerge. In this review, we discuss recent evidence of dramatic changes in the activity of an important signal transducer found to be profoundly affected by cell to cell adhesion, the signal transducer and activator of transcription-3 (Stat3). Direct cadherin engagement, growth of cells to postconfluence, or formation of multicellular aggregates were found to induce a striking increase in the levels of Stat3 activity, Rac1/Cdc42, and members of the IL6 receptor family in different settings. This activation was specific to Stat3, in that the levels of the extracellular signal regulated kinase (Erk1/2), a signal transducer often coordinately activated with Stat3 by a number of growth factors or oncogenes, remained unaffected by cell density. Density-dependent Stat3 activation may play a key role in survival, and could contribute to the establishment of cell polarity. It is clear that at any given time the total Stat3 activity levels in a cell are the sum of the effects of cell to cell adhesion plus the conventional Stat3 activating factors present.
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http://dx.doi.org/10.1139/o09-061DOI Listing
December 2009

Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration.

Exp Cell Res 2010 Mar 21;316(5):875-86. Epub 2009 Oct 21.

Departments of Microbiology and Immunology and Pathology and Molecular Medicine, and Queen's University Cancer Institute, Queen's University, Botterell Hall, Rm. 713, Kingston, Ontario, Canada K7L3N6.

Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac(V12)), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac(V12) with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac(V12)-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac(V12) is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac(V12) expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac(V12). The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac(V12), as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.
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http://dx.doi.org/10.1016/j.yexcr.2009.10.017DOI Listing
March 2010

Cadherin-cadherin engagement promotes cell survival via Rac1/Cdc42 and signal transducer and activator of transcription-3.

Mol Cancer Res 2009 Aug 11;7(8):1310-27. Epub 2009 Aug 11.

Department of Microbiology and Immunology, Department of Pathology and Molecular Medicine, and Cancer Research Institute, Queen's University, Ontario, Canada K7L 3N6.

Signal transducer and activator of transcription-3 (Stat3) is activated by a number of receptor and nonreceptor tyrosine kinases, whereas a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. In the present report, we show that Stat3 can also be activated through homophilic interactions by the epithelial (E)-cadherin. Indeed, by plating cells onto surfaces coated with fragments encompassing the two outermost domains of this cadherin, we clearly show that cadherin engagement can activate Stat3, even in the absence of direct cell-to-cell contact. Most importantly, our results also reveal for the first time an unexpected and dramatic surge in total Rac1 and Cdc42 protein levels triggered by cadherin engagement and an increase in Rac1 and Cdc42 activity, which is responsible for the Stat3 stimulation observed. Inhibition of cadherin interactions using a peptide, a soluble cadherin fragment, or genetic ablation induced apoptosis, points to a significant role of this pathway in cell survival signaling, a finding that could also have important therapeutic implications. (Mol Cancer Res 2009;7(8):1310-27).
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http://dx.doi.org/10.1158/1541-7786.MCR-08-0469DOI Listing
August 2009