Yuqi Jing

Antiangiogenic activity of 2-deoxy-D-glucose

Background During tumor angiogenesis, endothelial cells (ECs) are engaged in a number of energy consuming biological processes, such as proliferation, migration, and capillary formation. Since glucose uptake and metabolism are increased to meet this energy need, the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on in vitro and in vivo angiogenesis were investigated. Methodology/Principal Findings In cell culture, 2-DG inhibited EC growth, induced cytotoxicity, blocked migration, and inhibited actively forming but not established endothelial capillaries. Surprisingly, 2-DG was a better inhibitor of these EC properties than two more efficacious glycolytic inhibitors, 2-fluorodeoxy-D-glucose and oxamate. As an alternative to a glycolytic inhibitory mechanism, we considered 2-DGs ability to interfere with endothelial N-linked glycosylation. 2-DGs effects were reversed by mannose, an N-linked glycosylation precursor, and at relevant concentrations 2-DG also inhibited synthesis of the lipid linked oligosaccharide (LLO) N-glycosylation donor in a mannose-reversible manner. Inhibition of LLO synthesis activated the unfolded protein response (UPR), which resulted in induction of GADD153/CHOP and EC apoptosis (TUNEL assay). Thus, 2-DGs effects on ECs appeared primarily due to inhibition of LLOs synthesis, not glycolysis. 2-DG was then evaluated in two mouse models, inhibiting angiogenesis in both the matrigel plug assay and the LHBETATAG transgenic retinoblastoma model. Conclusions/Significance In conclusion, 2-DG inhibits endothelial cell angiogenesis in vitro and in vivo, at concentrations below those affecting tumor cells directly, most likely by interfering with N-linked glycosylation rather than glycolysis. Our data underscore the importance of glucose metabolism on neovascularization, and demonstrate a novel approach for anti-angiogenic strategies.

Tumor and vascular targeting of a novel oncolytic measles virus retargeted against the urokinase receptor.

Oncolytic measles virus (MV) induces cell fusion and cytotoxicity in a CD46-dependent manner. Development of fully retargeted oncolytic MVs would improve tumor selectivity. The urokinase-type plasminogen activator receptor (uPAR) is a tumor and stromal target overexpressed in multiple malignancies. MV-H glycoproteins fully retargeted to either human or murine uPAR were engineered and their fusogenic activity was determined. Recombinant human (MV-h-uPA) and murine (MV-m-uPA) uPAR-retargeted MVs expressing enhanced green fluorescent protein (eGFP) were rescued and characterized. Viral expression of chimeric MV-H was shown by reverse transcription-PCR and Western blot. In vitro viral replication was comparable to MV-GFP control. The receptor and species specificity of MV-uPAs was shown in human and murine cells with different levels of uPAR expression. Removal of the NH(2)-terminal fragment ligand from MV-uPA by factor X(a) treatment ablated the MV-uPA functional activity. Cytotoxicity was shown in uPAR-expressing human and murine cells. MV-h-uPA efficiently infected human endothelial cells and capillary tubes in vitro. I.v. administration of MV-h-uPA delayed tumor growth and prolonged survival in the MDA-MB-231 breast cancer xenograft model. Viral tumor targeting was confirmed by immunohistochemistry. MV-m-uPA transduced murine mammary tumors (4T1) in vivo after intratumor administration. MV-m-uPA targeted murine tumor vasculature after systemic administration, as shown by dual (CD31 and MV-N) staining of tumor capillaries in the MDA-MB-231 model. In conclusion, MV-uPA is a novel oncolytic MV associated with potent and specific antitumor effects and tumor vascular targeting. This is the first retargeted oncolytic MV able to replicate in murine cells and target tumor vasculature in a uPAR-dependent manner.

Role of Plasminogen Activator Inhibitor-1 in Urokinase's Paradoxical In Vivo Tumor Suppressing or Promoting Effects

Tumor proteases and inhibitors have been associated with paradoxical effects on tumor progression in preclinical and clinical settings. We previously reported that urokinase (uPA) overexpression delays tumor progression in mammary cancer. This study aimed to determine the role of plasminogen activator inhibitor-1 (PAI-1) on uPAs paradoxical in vivo effects. Using syngeneic murine models, we found that stable uPA overexpression promoted in vivo growth of colon tumors (MC-38) naturally expressing high PAI-1, whereas growth inhibition was observed in renal tumors (RENCA) expressing lower PAI-1 levels. In murine mammary carcinoma (4T1), uPA overexpression shifted the uPA/PAI-1 balance in favor of the protease, resulting in significantly reduced tumor growth and metastases in vivo. Conversely, increased tumor progression was observed in stable PAI-1 overexpressing 4T1 tumors as compared with uPA-overexpressing and control tumors. These effects were associated with downregulation of metastases promoting genes in uPA-overexpressing tumors, such as metalloproteinases, CXCL-1, c-Fos, integrin α-5, VEGF-A, PDGF-α, and IL-1β. In PAI-1–overexpressing tumors, many of the above genes were upregulated. PAI-1 overexpressing tumors had increased total and new tumor microvessels, and increased tumor cell proliferation, whereas the opposite effects were found in uPA-overexpressing tumors. Finally, PAI-1 downregulation led to significant inhibition of 4T1 tumor growth and metastases in vivo. In conclusion, uPAs dual effects on tumor progression occur in the context of its interactions with endogenous PAI-1 expression. Our studies uncover novel mechanisms of in vivo tumor control by modulation of the balance between tumor proteases and inhibitors, which may be exploited therapeutically. Mol Cancer Res; 10(10); 1271–81. ©2012 AACR.

2-Deoxy-Glucose Downregulates Endothelial AKT and ERK via Interference with N-Linked Glycosylation, Induction of Endoplasmic Reticulum Stress, and GSK3β Activation

Interference with endothelial cell metabolism is a promising, yet unexploited strategy for angiogenesis inhibition. We reported that the glucose analogue 2-deoxy-D-glucose (2-DG) inhibits angiogenesis at significantly lower concentrations than those required for tumor cytotoxicity. Here, we found that hypersensitivity to 2-DG in endothelial cells is not associated with enhanced drug uptake compared with tumor cells, but with time-dependent, endothelial-selective inhibition of AKT and ERK phosphorylation. Downregulation of these critical survival pathways is shown to be due to 2-DGs interference with N-linked glycosylation, leading to alterations in VEGFR2 (and downstream signaling) as well as induction of endoplasmic reticulum (ER) stress, GSK3β activation, and apoptosis. In vivo, periocular administration of 2-DG in LHBETATAG mice was associated with significant reduction of newly formed (CD105+) tumor capillaries, ER stress (GRP 78 expression), and endothelial apoptosis (TUNEL). These findings uniquely link N-linked glycosylation inhibition, ER stress, and ERK/AKT downregulation in endothelial cells, and provide a novel drug development strategy to overcome resistance mechanisms to currently available antiangiogenic agents. Mol Cancer Ther; 15(2); 264–75. ©2015 AACR.

In vivo anti-metastatic effects of uPAR retargeted measles virus in syngeneic and xenograft models of mammary cancer

Abstract The urokinase receptor (uPAR) plays a critical role in breast cancer (BC) progression and metastases and is a validated target for novel therapies. The current study investigates the effects of MV-uPA, an oncolytic measles virus fully retargeted against uPAR in syngeneic and xenograft BC metastases models. In vitro replication and cytotoxicity of MVs retargeted against human (MV-h-uPA) or mouse (MV-m-uPA) uPAR were assessed in human and murine cancer and non-cancer mammary epithelial cells. The in vivo effects of species-specific uPAR retargeted MVs were assessed in syngeneic and xenograft models of experimental metastases, established by intravenous administration of luciferase expressing 4T1 or MDA-MD-231 cells. Metastases progression was assessed by in vivo bioluminescence imaging. Tumor targeting was evaluated by qRT-PCR of MV-N, rescue of viable viral particles, and immunostaining of MV particles in lungs from tumor bearing mice. In vitro, MV-h-uPA and MV-m-uPA selectively infected, replicated, and induced cytotoxicity in cancer compared to non-cancer cells in a species-specific manner. In vivo, MV-m-uPA delayed 4T1 lung metastases progression and prolonged survival. These effects were associated with identification of viable viral particles, viral RNA, and detection of MV-N by immunostaining from lung tissues in treated mice. In the human MDA-MB-231 metastases model, intravenous administration of MV-h-uPA markedly inhibited metastases progression and significantly improved survival, compared to controls. No significant treatment-related toxicity was observed in treated mice. The above preclinical findings strongly suggest that uPAR retargeted measles virotherapy is a novel and feasible systemic therapy strategy against metastatic breast cancer.

In vivo safety, biodistribution and antitumor effects of uPAR retargeted oncolytic measles virus in syngeneic cancer models

The urokinase receptor (uPAR) is a clinically relevant target for novel biological therapies. We have previously rescued oncolytic measles viruses fully retargeted against human (MV-h-uPA) or murine (MV-m-uPA) uPAR. Here, we investigated the in vivo effects of systemic administration of MV-m-uPA in immunocompetent cancer models. MV-m-uPA induced in vitro cytotoxicity and replicated in a receptor-dependent manner in murine mammary (4T1) and colon (MC-38 and CT-26) cancer cells. Intravenous administration of MV-m-uPA to 4T1 tumor-bearing mice was not associated with significant clinical or laboratory toxicity. Higher MV-N RNA copy numbers were detected in primary tumors, and viable viral particles were recovered from tumor-bearing tissues only. Non-tumor-bearing organs did not show histological signs of viral-induced toxicity. Serum anti-MV antibodies were detected at day 14 of treatment. Immunohistochemistry and immunofluorescence studies confirmed successful tumor targeting and demonstrated enhanced MV-m-uPA-induced tumor cell apoptosis in treated compared with control mice. Significant antitumor effects and prolonged survival were observed after systemic administration of MV-m-uPA in colon (CT-26) and mammary (4T1) cancer models. The above results show safety and feasibility of uPAR targeting by an oncolytic virus, and confirm significant antitumor effects in highly aggressive syngeneic immunocompetent cancer models.

Abstract 1734: In vivo immunomodulatory effects by uPAR-retargeted oncolytic Measles virus

Background: Oncolytic viruses offer an advantage over other treatment options, as they both induce cytotoxicity as well as immune modulation. Oncolytic measles virus (MV) is a safe and promising oncolytic viral vector that is being tested in early clinical trials for treatment of cancer. While the oncolytic activity of MV has been clearly defined, the virus9 in vivo immunomodulatory effects have not been well established, as CD46 (the endogenous MV receptor) is expressed in human, but not murine cells. We have developed MV-m-uPA, a fully retargeted MV that can selectively target mouse tissues in a m-uPAR dependent manner. The goals of this study are to characterize the in vivo immunomodulatory effects of MV-m-UPA. Methods: Immunocompetent Balb/c mice were injected subcutaneously with CT-26 tumors. When tumor growth reached 4-5 mm, mice were treated with either vehicle control or MV-m-UPA via tail vein (IV) or intratumor (IT) every other day for two doses. Tumors were resected and changes in the immune microenvironment were analyzed by flow cytometry at days 5 and 10 after treatment. Results: IV and IT administration of MV-m-UPA induced differential, time-dependent changes of components of innate and adaptive immunity. At early time points (day 5), intravenous virus treatment was associated with increases in MDSCs and NK cells, while the opposite effects were observed after IT administration. Analysis of CD8 + T cell subpopulations showed that IV treatments led to a decrease in TIM3 + /PD1 + (exhausted) CD8 + T cells, while TIM3 - /PD1 - CD8 + cells increased after IV administration. The opposite effect was observed after IT administration. While both CD4 + T helper and FOXP3 + CD4 + cells increased after IV treatments, no major changes occurred after IT treatments. Interestingly, while a significant decrease in TAM 2 cells was observed at days 5 and 10 in both groups, TAM 1 cells increased only in the IV group at day 10. Conclusions: In summary, IV MV-m-uPA administration induces overall decreases in TAM 2 and MDSCs, while NK and TAM 1 cells increase, changes that may be associated with tumor control. On the other hand, FOXP3 + cells increase, which may be a mechanism of immune suppression after IV administration. IT administration is associated with a decrease in MDSCs, TAM 2, as well as a decrease in FOXP3 + CD4 + cells, while no significant changes were observed in TAM 1 cells. The above results demonstrate for the first time the dynamic changes that occur within the immune microenvironment upon administration an oncolytic measles virus in a fully immunocompetent cancer model. These findings may help better understand positive and negative changes as a result of MV infection and may lead to identification of rational combinations of viruses with selected immunotherapeutic agents. Citation Format: Natasha K. Khatwani, Yuqi Jing, Valery Chavez, Jaime Merchan. In vivo immunomodulatory effects by uPAR-retargeted oncolytic Measles virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1734.

Molecular Effects of Stromal-Selective Targeting by uPAR-Retargeted Oncolytic Virus in Breast Cancer

The tumor microenvironment (TME) is a relevant target for novel biological therapies. MV-m-uPA and MV-h-uPA are fully retargeted, species-specific, oncolytic measles viruses (MV) directed against murine or human urokinase receptor (PLAUR/uPAR), expressed in tumor and stromal cells. The effects of stromal-selective targeting by uPAR-retargeted MVs were investigated. In vitro infection, virus-induced GFP expression, and cytotoxicity by MV-h-uPA and MV-m-uPA were demonstrated in human and murine cancer cells and cancer-associated fibroblasts in a species-specific manner. In a murine fibroblast/human breast cancer 3D coculture model, selective fibroblast targeting by MV-m-uPA inhibited breast cancer cell growth. Systemic administration of murine-specific MV-m-uPA in mice bearing human MDA-MB-231 xenografts was associated with a significant delay in tumor progression and improved survival compared with controls. Experiments comparing tumor (MV-h-uPA) versus stromal (MV-m-uPA) versus combined virus targeting showed that tumor and stromal targeting was associated with improved tumor control over the other groups. Correlative studies confirmed in vivo viral targeting of tumor stroma by MV-m-uPA, increased apoptosis, and virus-induced differential regulation of murine stromal genes associated with inflammatory, angiogenesis, and survival pathways, as well as indirect regulation of human cancer pathways, indicating viral-induced modulation of tumor–stroma interactions. These data demonstrate the feasibility of stromal-selective targeting by an oncolytic MV, virus-induced modulation of tumor–stroma pathways, and subsequent tumor growth delay. These findings further validate the critical role of stromal uPAR in cancer progression and the potential of oncolytic viruses as antistromal agents. Implications: The current report demonstrates for the first time the biological, in vitro, and in vivo antitumor and molecular effects of stromal selective targeting by an oncolytic virus. Mol Cancer Res; 15(10); 1410–20. ©2017 AACR.

Abstract 3745: Tumor fibroblast targeting via uPAR retargeted measles virus: In vitro and in vivo effects

Tumor stromal cell components, in particular cancer associated fibroblasts, play an important role in cancer progression. Few studies have focused on stromal fibroblast targeting by oncolytic viruses. The urokinase receptor (uPAR) is a clinically and biologically validated target, which is overexpressed in tumor and stromal cells compared to non-cancer tissues. MV-h-uPA and MV-m-uPA are fully retargeted oncolytic measles viruses directed against human and murine uPAR, respectively, which have shown in vitro and in vivo safety and antitumor effects. Species specific retargeted viral vectors allow to dissect the specific effects of the viruses on murine vs. human tissues in xenograft models. Our aim is to characterize the in vitro and in vivo effects of stromal targeting by oncolytic measles virus via uPAR, with a focus on tumor fibroblasts. In vitro, MV-h-uPA and MV-m-uPA preferentially infected and induced cytotoxicity in human cancer associated fibroblasts (CAF 19, CAF 23) as well as murine fibroblasts (3T3), compared to non-tumorigenic fibroblasts. Murine-murine and human-human fibroblast to cancer cell viral transfer via heterofusion was observed after fibroblast infection by species specific MV-uPA, in breast, colon and renal cancer models, while no viral transfer was observed between cells of different species. In vivo, systemic administration of the murine uPA retargeted virus (MV-m-uPA) significantly decreased tumor progression and prolonged survival in a human breast cancer xenograft model (MDA-MD231), where the host stroma expresses murine uPAR (target of MV-m-uPA). Tumor studies revealed induction of apoptosis (TUNEL assay), while no significant effects on cancer cell proliferation was observed. Dual staining for measles virus nucleocapsid proteins and fibroblasts markers demonstrated viral infection of fibroblasts in treated tumors. Gene expression studies using murine as well as human specific arrays were performed to characterize the effects of stromal targeting by the murine retargeted virus on murine stroma as well as the indirect effects on human cancer cells in vivo. In conclusion, for the first time our results show feasibility and antitumor effects of stromal fibroblast targeting by oncolytic measles virus via uPAR and demonstrate that stromal fibroblasts are viable targets for oncolytic virotherapy. Studies characterizing other stromal cell components and evaluation of the molecular changes in the tumor stroma as result of viral infection are underway. Citation Format: Yuqi Jing, Julia Zaias, Jaime Merchan. Tumor fibroblast targeting via uPAR retargeted measles virus: In vitro and in vivo effects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3745.

Abstract 3545: Stromal selective targeting by uPAR retargeted oncolytic measles virus inhibits breast cancer progression

Stromal cells in the tumor microenvironment play an important role in breast cancer progression. Strategies directed against stromal cell components have been investigated in the preclinical and clinical setting. However, few studies have focused on stromal targeting by oncolytic viruses. The urokinase receptor (uPAR) is overexpressed in tumor and stromal cells, and plays a critical role in tumor progression. The aim of this study is to evaluate the antitumor effects of stromal retargeted oncolytic measles virus via urokinase receptor. We rescued and characterized fully retargeted oncolytic measles viruses against human (MV-h-uPA) and murine (MV-m-uPA) urokinase receptor (uPAR). In vitro, MV-m-uPA and MV-h-uPA infected and induced cytotoxicity to human (MDA-MB231, T47D and MCF-7) and murine (4T1) mammary cancer cells in a species specific manner. Efficient MV-m-uPA infection and replication were demonstrated in murine NIH-3T3 mouse fibroblast cells and MS1 murine endothelial cells. Selective infection of murine fibroblasts by the murine uPAR specific MV-m-uPA led to significant inhibition of human breast cancer proliferation in an in vitro 3-D collagen co-culture model of tumor-stromal interactions. To further validate the potential of stromal uPAR targeting as a therapeutic strategy, the effects of MV-uPA were assessed in a human breast cancer xenograft model (MDA-MD231), where tumor cells express human uPAR (target of MV-h-uPA) and the host stroma expresses murine uPAR (target of MV-m-uPA). Tumor bearing mice were treated with MV-h-uPA, MV-m-uPA or combination (MV-h-uPA and MV-m-uPA) by IV administration (3 doses). Surprisingly, MV-m-uPA (selectively targeting mouse uPAR) was associated with a measurable delay in tumor progression while on treatment, as well as prolongation in survival. Combination therapy (MV-m-uPA and MV-h-uPA) was associated with the best antitumor effects in vivo. To further characterize the in vivo effects of stromal uPAR targeting, repeat experiments were performed where tumor bearing mice received prolonged treatment with MV-m-uPA (12 treatments). This approach was shown to be safe and resulted in a significant delay in tumor progression and marked prolongation of survival. These effects were associated with the identification of viable viral particles and viral RNA from MDA-MB231 tumor xenograft treated with MV-m-uPA. In conclusion, our results demonstrate for the first time that oncolytic viral targeting of tumor stroma is feasible and associated with in vivo antitumor effects. These findings further validate the critical role of stromal uPAR in cancer progression. Citation Format: Yuqi Jing, Marcela Toro Bejarano, Krisztina Kovacs, Jaime Merchan. Stromal selective targeting by uPAR retargeted oncolytic measles virus inhibits breast cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3545. doi:10.1158/1538-7445.AM2015-3545