Dzung H. Dinh

Abstract 5187: Urokinase plasminogen activator receptor (uPAR) and integrin α5β1 regulate plasminogen activator inhibitor-1 (PAI-1) activity in medulloblastoma cells

The urokinase plasminogen activator (uPA) system is a dynamic extracellular protease system that regulates both proteolytic and non-proteolytic events associated with cancer progression. In earlier studies, we have demonstrated that radiation-induced cell adhesion was associated with uPAR expression and downregulation of uPAR effectively inhibited radiation-induced cell adhesion of medulloblastoma cells. Herein, we further investigated the impact of uPAR downregulation on plasminogen activator inhibitor (PAI-1), a key regulator component of the uPA system, and its role in cell adhesion. With radiation treatment, the levels of uPAR and PAI-1 increased in D283 and UW228 cells. Surprisingly, knockdown of uPAR elevated the levels of PAI-1. The increase in PAI-1 levels in uPAR-knocked down cells was regulated by activation of cJUN/CREB signaling molecules. Moreover, studies show that the reduced interaction between uPAR and integrin α5β1 plays a critical role in PAI-1 release into the extracellular matrix, thereby enhancing the cell detachment process. Incubation of medulloblastoma cells with Tiplaxtinin-PAI-039, a potent PAI-1 activity inhibitor, increased the association of uPAR with integrin α5β1 and cell adhesion. Interestingly, prolonged inhibition of PAI-1 activity adversely affected cell viability; we were able to revert this effect by exogenously supplementing cells with recombinant PAI-1. Moreover r-PAI-1 showed no significant effect on cell adhesion whereas it increased the association of uPAR with integrin α5β1. Expression of full-length (FL) uPAR elevated uPA and PAI-1 levels both at the transcriptional and the translational levels. These increased PAI-1 levels play a pivotal role in regulating uPA activity by binding and translocating integrin α5β1 towards the uPA/uPAR complex. Taken together, the results of the present study lead us to conclude that levels of uPAR and integrin α5β1, and their association on the cell surface dictate the function of PAI-1 to maintain extracellular homeostasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5187. doi:1538-7445.AM2012-5187

Abstract 3376: Cord blood stem cells revert EMT to MET in glioma stem cells by downregulating synergistic transcriptional activation of Sox2 and Twist1

The dynamic nature of cancer stem cells that underlie metastasis or their ability to switch between different cellular identities, as in epithelial to mesenchymal transition and mesenchymal to epithelial transition has profound implications for cancer therapy. The functional relationship between molecules involved in cancer cell stemness and metastasis is not clear. In this regard, our studies on human glioblastoma tissue grade IV specimens showed significant expression of Twist1 and Sox2, known mesenchymal and stemness related markers, respectively, indicating their association with glial tumorigenesis and metastasis. The glioma stem cells obtained from CD133 + cells demonstrated increased expression of Twist1 and Sox2 accompanied by significant increase in the mesenchymal markers such as N-cadherin, vimentin and β-catenin. Our studies on glioma stem cells treatment with human umbilical cord blood derived mesenchymal stem cells, showed down regulation of Twist1 and Sox2 proteins, apart from other mesenchymal stem cell markers. Based on the in vitro experiments and in vivo intracranial xenograft mouse model studies we elucidated the potential therapeutic role of hUCBSC in suppressing glioma cancer stemness by the induction of mesenchymal to epithelial transition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3376. doi:1538-7445.AM2012-3376

Abstract 1118: Protease-integrin regulates mitochondrial biogenesis in glioma xenograft cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Various studies have described mitochondrial dysfunction and its association with several diseases, including cancer. Regulation of mitochondrial biogenesis via mitochondrial-nuclear genome encoded protein signaling has been identified in recent literature. In the present study, we explored whether matrix metalloproteinase-9 (MMP-9) shRNA mediates mitochondrial biogenesis and alters glioma proliferation. We found that MMP-9 shRNA initiated mitochondrial biogenesis in glioma cells by increasing the expression of mitochondrial transcription factor (mtTFA). MMP-9 silencing elevated nuclear respiratory factor-1 (NRF1) and -2 (NRF2), which are involved in transcription regulation of mtTFA. Mitochondrial localization of mtTFA was more evident in MMP-9-treated glioma cells as compared to untreated cells. In addition, MMP-9 suppression significantly increased the binding of mtTFA to light strand and heavy strand promoter of mitochondrial genome as determined by mitochondrial chromatin immunoprecipitation assay. MMP-9 transcriptional suppression decreased the expression of phospho Akt and PI3K while increasing PTEN expression in glioma xenograft cells. We also show that dominant negative Akt increased mtTFA, NRF1 and NRF2, and decreased PI3K, integrin αv and integrin β3. Further, MMP-9 and Akt suppression increased the nuclear and mitochondrial expression of mtTFA. As indicated by increased copy number of displacement (D) loop, mitochondrial replication was observed in both MMP-9- and Akt-downregulated glioma cells. As confirmed by immunoprecipitation analysis, interaction of αvβ3 integrin with MMP-9 decreased. Studies with functional blocking of αvβ3 integrin indicated elevation of mtTFA, a decrease in phospho Akt, and overexpression of D-loop; these data indicate MMP-9 mediates mitochondrial biogenesis via αvβ3 integrin-Akt/PTEN, which led to increased mtTFA expression. Earlier studies from our lab also showed apoptosis induction in these glioma cells by MMP-9 shRNA. Moreover, 4910 and 5310 glioma xenograft tissue sections from mice treated with MMP-9 shRNA showed reduced expression of phospho Akt, and elevated PTEN and mtTFA levels. Decreased co-localization of αvβ3 integrin and MMP-9 was associated with MMP-9-suppressed tumor sections. Further, RT-PCR and western blot analyses showed increased expression of proteins involved in mitochondrial biogenesis encoded both by mitochondrial and nuclear genome in MMP-9 shRNA-treated glioma cells. Elevated levels of reactive oxygen species and the results of the FACS analysis show increased mitochondrial organelle stain in MMP-9-silenced glioma cells. Taken together, these data provide new insight into MMP-9-mediated glioma proliferation inhibition by mitochondrial biogenesis induced cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1118. doi:1538-7445.AM2012-1118

Abstract 2218: Radiation-induced hypomethylation triggers urokinase plasminogen activator (uPA) transcription in meningioma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Our previous studies have shown the role of radiation-induced urokinase plasminogen activator (uPA) expression in meningioma tumor progression. In the present study, we investigated whether modulation of DNA methylation profiles could regulate uPA expression. Radiation treatment induced hypomethylation in meningioma cells with a decrease in DNMT1 and MBD expression. However, oxidative damage by H2O2 or pretreatment of irradiated cells with NAC did not show any influence on these proteins, thereby indicating a radiation-specific change in the methylation patterns among meningioma cells. Further, we identified that hypomethylation is coupled to an increase in uPA expression in these cells. Azacytidine treatment induced a dose-dependent surge of uPA expression while pretreatment with Sodium butyrate inhibited the radiation-induced uPA expression; these results complement our prior findings. Methylation-specific PCR on bisulfite treated genomic DNA revealed a diminished methylation of uPA promoter in irradiated cells, which supports the role of epigenetics in uPA gene regulation. Transfection with shRNA expressing plasmids targeting CpG islands of the uPA promoter showed a marked decline in uPA expression with subsequent decreases in invasion and proliferation of meningioma cells. Further, radiation treatment is coupled to the recruitment of SP1 transcription into the nuclear extracts, which was abrogated by shRNA treatment. Our experiments to study the signaling events demonstrated the activation of MEK-ERK in radiation treated cells while U0126 (MEK/ERK inhibitor) blocked hypomethylation, recruitment of SP1, and uPA expression. In agreement with our in vitro data, low DNMT1 levels and high uPA were found in intracranial tumors given radiation treatment as compared to untreated tumors. In conclusion, our data suggest radiation-mediated hypomethylation triggers uPA expression in meningioma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2218. doi:1538-7445.AM2012-2218

Abstract 2085: MMP-2 knockdown leads to anoikis and inhibits migration and invasion by attenuating αvβ3/EGFR/Pak4 survival signaling in human glioma xenograft cells in vitro and in vivo

MMP-2 plays a key role in the degradation of the extracellular matrix and thereby enhances the proliferative, invasive and metastatic potential of cancer cells. MMP-2 knockdown using siRNA (pM) in human glioma xenograft cell lines, 4910 and 5310, sensitized cells to anoikis associated with cleavage of PARP, and caspases 8 and 3. pM-inhibited cell adhesion, invasion and migration correlated with attenuation of EGFR, c-Src and Pak4 phosphorylation. High expression of Pak4 in human glioma cell lines and tumor tissues implicated its essential role in increasing glioma malignancy. Co-immunoprecipitation studies implied that MMP-2 directly interacts by complex formation with EGFR, p130CAS and Pak4 along with αvβ3 integrin and pM treatment disrupted this direct binding. Vitronectin-mediated αvβ3 activation and EGF and rhMMP-2 supplementation counteracted pM-inhibited, Pak4-mediated migration and invasion. Conversely, αvβ3 blocking antibody and ErbB-2/EGFR inhibitor (GW2974) further reduced pM-inhibited, Pak4-mediated invasiveness and migration, thereby suggesting a role of αvβ3 and EGFR in the regulation of Pak4 survival signaling. Individual treatments of pM and Pak4.si led to considerable anoikis in both 4910 and 5310 cells and simultaneous co-depletion MMP-2 and Pak4 resulted in robust increase in anoikis levels. In vivo orthotopic experiments in nude mice revealed decreased tumor size with pM treatment. Immunofluorescence analysis of pSV-tumor sections showed high expression and co-localization of MMP-2/αvβ3, which decreased upon treatment with pM along with significant decrease in phospho-EGFR, phospho-Pak4, Bcl2 and Bcl-xL levels. Our results imply a possible crosstalk between MMP-2 and Pak4 and suggest the potential of targeting both MMP-2 and Pak4 as a future therapeutic approach in glioma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2085. doi:1538-7445.AM2012-2085

Abstract 3322: Downregulation of XIAP by cord blood stem cells is associated with inhibition of heat shock protein 90 in glioblastoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Heat shock protein 90 (HSP90) is highly expressed under stressful conditions in eukaryotic cells showing chaperone activity including the suppression of protein aggregation and signaling pathways. HSP90 is upregulated in various human tumors, including glioblastoma, and targeting its function might provide new therapeutic opportunities. Previously, we have shown that human umbilical cord blood-derived mesenchymal stem cells (hUCBSC) downregulate XIAP in glioblastoma cells in vitro and in vivo. In the present study, we report that downregulation of XIAP by hUCBSC is associated with inhibition of HSP90. Under in vivo conditions, hUCBSC induce apoptosis by downregulation of XIAP in which XIAP migrates from the cytosol to the nucleus in mice brain xenografts. Immunoprecipitation experiments proved that SMAC is associated with Cytochrome C and XIAP in the mitochondria, indicating mitochondrial apoptosis. Simultaneously, cytosolic HSP90 is downregulated and co-localized with TNFR2 in hUCBSC-treated tumors, proving that TRAIL-mediated apoptosis is taking place in hUCBSC-treated tumor xenografts. Also, expression of mitochondrial HSP90 (TRAP-1) is downregulated in glioma cells. The downregulation of XIAP by hUCBSC is shown to be under the transcriptional control of SP1 under both in vitro and in vivo conditions. These results have been confirmed by using shRNA for XIAP. Taken together, these results prove that hUCBSC are efficient in the inhibition of HSP90, and hUCBSC have the potential to be therapeutic tools for the treatment of glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3322. doi:10.1158/1538-7445.AM2011-3322

Abstract 576: Gadd45a sensitizes medulloblastoma cells to irradiation and modulates the cellular switch from migratory to stationary cell phase by downregulating MMP-9

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Gadd45a protein is induced in medulloblastoma cell lines by treatment with ionizing radiation (IR), which requires p53 stabilization. The role of Gadd45a in IR-induced G2-M arrest of medulloblastoma cells is demonstrated by its increased binding to Cdc2, thereby inactivating Cdc2 kinase activity. Knockdown of Gadd45a alleviates G2-M blockage and results in decreased binding of Gadd45a to Cdc2. Further, the anti-tumorigenic role of Gadd45a is mediated by the negative regulation of β-catenin and its nuclear translocation, which might decrease the β-catenin/LEF-1-mediated transactivation of cell invasion proteins, such as MMP-9, examined in the present study. Overexpression of Gadd45a protein with IR treatment resulted in decreased nuclear levels of β-catenin, increased cytoplasmic levels of p-β-catenin, and increased distribution of β-catenin on membrane E and N-cadherins. The IR-treated control cells showed contrary results with demonstrated loss of E-cadherin, gain of N-cadherin, and inhibition of β-catenin binding to membrane cadherins as well as increased nuclear translocation of β-catenin. Zymography studies demonstrate the negative regulation of IR-induced MMP-9 activity by Gadd45a overexpression. Wound healing and Matrigel invasion assays confirmed the anti-tumorigenic and anti-metastatic role of Gadd45a in medulloblastoma cell lines. Furthermore, siRNA-mediated knockdown of MMP-9 results in prominent expression levels of Gadd45a protein in animal tumors. Thus, we confirm that overexpression of Gadd45a in combination with ionizing radiation has therapeutic implications by preventing recurrence of MMP-9-mediated cancer cell invasion and effectively containing cells in a static phase by maintaining CCA/CCI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 576. doi:10.1158/1538-7445.AM2011-576

Abstract 2938: Inhibition of PI3K/AKT and MEK/ERK pathways act independently on p27 upregulation induced by downregulation of cathepsin B and uPAR in glioma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cathepsin B and urokinase plasminogen activator receptor (uPAR) are overexpressed in gliomas. Deregulation of the G1 phase cell cycle machinery is a common feature of cancers. The abundance of cyclin-dependent kinase inhibitor p27Kip1 (p27) during the cell cycle determines whether cells will proliferate or become quiescent. In the present study, we carried out transfections using shRNA against cathepsin B and uPAR. We assessed cell cycle arrest using FACS analysis and used Western blot analysis to detect expression of ERK, AKT, p27, cyclin E, cyclin D, CDK4, CDK2, EGFR, β1 integrins, and the pocket proteins (p-Rb, p107 and p130). Specific inhibitors like Wortmanin (10 µM) and U0126 (10 µM) were used to further determine the roles of the AKT and ERK pathways. We used immunoprecipitation analysis to detect the respective interactions between EGFR and β1, p27 and CDK4, and p27 and CDK2. CHIP analysis was used to check for the recruitment of transcription factors FOXO3a, E2F1 and AP-1 to the p27 promoter. The results of the present study show that shRNA treatment efficiently downregulated expression of cathepsin B and uPAR and induced G0/G1 arrest through upregulation of p27. Downregulation of both p-ERK and p-AKT downstream of EGFR and β1 are involved, but independently, in p27 upregulation. Multiple transcription factors (namely, AP-1, E2F1 and FOXO3a) were involved in upregulation of p27 as determined by luciferase expression under the influence of promoter regions to which these transcription factors bind individually. Also, cathepsin B and uPAR downregulation reduced tumor growth and increased p27 nuclear expression in vivo. In summary, the reduction of p-ERK and AP-1, the increased expression of E2F1 and FOXO3a, and the reduced association of cyclin D-CDK4 and cyclin E-CDK2 complexes contributed to p27 upregulation as well as G0/G1 arrest induced by downregulation of cathepsin B and uPAR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2938. doi:10.1158/1538-7445.AM2011-2938

Abstract 2389: uPA and uPAR siRNA inhibits angiogenesis by blocking nuclear localization of angiogenin and angiopoietin1 signaling in glioblastoma cell lines

Transcriptional inactivation of uPA and uPAR system in the glioblastoma cell line SNB19 inhibited angiogenesis both in vitro and in vivo. To decipher this mechanism and extend our studies to other glioblastoma cell lines, U87MG and U87 SPARC-overexpressing cell lines were subjected to uPA, uPAR and U2 (bicistronic construct against uPA and uPAR) downregulation by siRNA. In the present study, the bicistronic construct against uPA and uPAR caused significant inhibition of angiogenesis by in vitro angiogenesis assay and in vivo dorsal skin fold chamber models in both cell lines. Increased apoptosis was seen in siRNA downregulated gliomablastoma cells when compared with control cells by FACS analysis. MTT assay revealed significant inhibition of proliferation by U2 siRNA treatment in both cell lines. Transfection with siRNA against uPA and uPAR in co-cultures of glioma cells with endothelial cells showed significant inhibition of angiopoeitin1, IL-6, GRO and GM-CSF while SPARC-overexpressing U87 cells had decreased secretion of angiogenin, GRO, MCP-1, PDGFBB, VEGF and angiopoietin1 by protein array analysis. Bicistronic construct showed decreased levels of angiogenin by ELISA and Western blot in endothelial cells and in cocultures with cancer cells. Transcriptional inactivation of uPA and uPAR showed significant downregulation of angiogenin-induced phosphorylation of ERK, JNK and Akt signaling in endothelial cells. Nuclear translocation of angiogenin in semi-confluent human umbilical vein endothelial cells treated with conditioned medium from siRNA-transfected U87 SPARC-overexpressing cells was inhibited as shown by Western blot and immunofluorescence analyses. Conditioned medium from U87MG and U87 SPARC-overexpressing cells transfected with uPA, uPAR and U2 siRNA plasmids significantly inhibited the phosphorylation of Tie2 receptor in endothelial cells. uPA and uPAR downregulation significantly downregulated MMP-2 levels by gelatin zymography in co-cultures and in single cultures of cancer cells. Matrix metalloproteinase array analysis showed significant inhibition of MMP-3 in co-cultures of U2-transfected SPARC-overexpressing glioma cells. Overall, our results suggest that the simultaneous downregulation of uPA and uPAR inhibits angiogenesis by inhibition of nuclear localization of angiogenin, angiopoeitin 1 signaling and MMP-2 downregulation in endothelial cells. In summary, these results identify novel mechanisms by which the bicistronic construct against uPA and uPAR inhibits angiogenesis, thereby suggesting the potential use of U2 as an anti-angiogenic agent against glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2389.

Abstract 3323: Induction of apoptosis in glioma cells by downregulation of X-linked inhibitor of apoptosis protein (XIAP)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC XIAP (X-linked inhibitor of apoptosis protein) is one of the most important members of the family of apoptosis inhibitors. XIAP blocks apoptosis by inhibiting both the initiator (caspase 9) and effector (caspase 3) caspases, and therefore, prevents cell death induced by various triggers. One mechanism through which tumor cells are believed to acquire resistance to apoptosis is by overexpression of XIAP. When XIAP is overexpressed, cancer cells are rendered resistant to apoptosis, both intrinsically and in response to chemotherapy and radiotherapy. It is upregulated in various malignancies, including human glioblastoma. It promotes invasion, metastasis, growth, and survival of malignant cells. In order to downregulate XIAP in glioma cells, we used human umbilical cord blood mesenchymal stem cells (hUCB) and siRNA of XIAP (siXIAP). We observed the effect of both hUCB and siXIAP on two malignant glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310). In either case, proliferation of the glioma cells was significantly inhibited. Both treatments increased cytotoxicity of glioma cells and led to cell death. However, cytotoxicity is more significant in siXIAP treatments compared to hUCB cocultures. Both hUCB and siXIAP induced TNF-alpha-mediated apoptosis in glioma cells, which was confirmed by TUNEL assay, FACS analyses and immunoblotting. Apoptosis is characterized by loss of mitochondrial membrane potential and upregulation of mitochondrial apoptotic protein BAD. Cell death of glioma cells is marked by downregulation of Akt and phospho-Akt molecules. Taken together, our results indicate the therapeutic potential of XIAP and hUCB in treating malignant gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3323.