Meena Gujrati

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 3351: Cord blood stem cells inhibit epidermal growth factor receptor translocation to mitochondria in glioblastoma

Overexpression of EGFR is one of the most frequently diagnosed genetic aberrations of glioblastoma multiforme (GBM). EGFR signaling is involved in diverse cellular functions and is dependent on the type of preferred receptor complexes. EGFR translocation to mitochondria has been reported recently in different cancer types. However, mechanistic aspects of EGFR translocation to mitochondria in GBM have not been evaluated to date. In the present study, we analyzed the expression of EGFR in GBM-patient derived specimens using immunohistochemistry, reverse-transcription based PCR and Western blotting techniques. In clinical samples, EGFR co-localizes with FAK in mitochondria. We evaluated this previous observation in standard glioma cell lines and in vivo mice xenografts. We further analyzed the effect of human umbilical cord blood stem cells (hUCBSC) on the inhibition of EGFR expression and EGFR signaling in glioma cells and xenografts. Treatment with hUCBSC inhibited the expression of EGFR and its co-localization with FAK in glioma cells. Also, hUCBSC inhibited the co-localization of activated forms of EGFR, FAK and c-Src in mitochondria of glioma cells and xenografts. In addition, hUCBSC also inhibited EGFR signaling mechanisms in glioma cells both in vitro and in vivo. We have shown that hUCBSC treatments inhibit phosphorylation of EGFR, FAK and c-Src forms. Our findings associate EGFR expression and its localization to mitochondria with specific biological functions in GBM cells and provide relevant preclinical information that can be used for the development of effective hUCBSC-based therapies. 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 3351. doi:1538-7445.AM2012-3351

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, FLnnGadd45a 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.nnCitation 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 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, DCnnXIAP (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.nnCitation 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.

Abstract 4986: Urokinase plasminogen activator (uPA) interacts with nuclear HOXA5 and inhibits p53 expression in glioma stem cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnUrokinase-type plasminogen activator (uPA) participates in diverse physiological processes, including glioma progression. HOXA5 is a known regulator of p53 expression, and overexpression of HOXA5 has been shown to increase p53 expression. In this study, we have demonstrated that uPA not only translocates to the nucleus but also interacts with the transcription factor HOXA5. We used CD133- and CD44-enriched U87 human glioma cells and 4910 human glioma xenograft cells, which exhibit cancer stem cell properties. Nuclear localization of uPA was determined by western blot analysis of nuclear fractions and by immunocytochemistry. Glioma stem cells showed increased nuclear localization of uPA as compared to normal glioma cells. To determine the in vitro interaction of uPA with HOXA5, we initially used membrane immobilized transcription factors incubated with purified uPA protein and looked for strong protein-protein interactions. We observed that uPA binds preferentially to HOXA5 transcription factor. Subsequently, we immunoprecipitated uPA or HOXA5 from nuclear fractions of U87 and 4910 normal and stem cells and tested for co-precipitation of HOXA5 or uPA respectively. Again, the results showed that uPA binds preferentially to nuclear HOXA5. To determine the effect of uPA or HOXA5 downregulation on these glioma cells, we used RNAi-expressing plasmids targeting uPA or HOXA5. The results showed that downregulation of uPA alone induced overexpression of p53 in both normal and glioma stem cells. Glioma stem cells downregulated for uPA showed at least a 5-fold increase in p53 expression when compared to uPA downregulated normal glioma cells. Downregulation of HOXA5 alone did not show significant change in expression levels of p53; however, simultaneous downregulation of uPA and HOXA5 did not show change in expression levels of p53. To determine whether uPA interacts directly with HOXA5 binding sequences, we performed an electromobility shift assay (EMSA). We observed that uPA does not bind to HOXA5 sequences but rather forms a complex with HOXA5 bound to DNA. In vivo studies showed that subcutaneous tumors injected intratumorally with siRNA-expressing plasmids targeting uPA or HOXA5 either singly or simultaneously showed strong p53 expression in uPA downregulated tumors with no significant change observed in other treatment conditions. Taken together, these results demonstrate that uPA behaves as a negative regulator of p53 expression in glioma cells.nnNote: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.nnCitation 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 4986.

Abstract 2286: SPARC stimulates neuronal differentiation of medulloblastoma cells via the IL-6/STAT3 pathway

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnSecreted Protein Acidic and Rich in Cysteine (SPARC), a 37-kDa glycoprotein, participates in the regulation of morphogenesis and cellular differentiation through its modulation of cell-matrix interactions. We have reported previously that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we show that the ectopic expression of SPARC induces neuronal differentiation in medulloblastoma cells. Adenovirus-mediated expression of SPARC cDNA (Ad-DsRed-SP) elevated the expression of neuronal markers NeuN, Nestin and MAP-2 in medulloblastoma cells. SPARC expression decreased STAT3 phosphorylation and constitutive expression of STAT3 reversed SPARC-mediated neuronal differentiation. Moreover, our results show that SPARC expression not only inhibited interleukin (IL)-6 but also attenuated IL-6-mediated STAT3 phosphorylation in medulloblastoma cells. Taken together, our results suggest that SPARC induced neuronal differentiation of medulloblastoma tumor cells through its inhibitory effect on IL-6-mediated STAT3 signaling. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed increased staining for the neuronal markers NeuN, Nestin and MAP-2. Overall, our results demonstrate that SPARC promotes neuronal differentiation and inhibits tumor growth, thereby further supporting the potential use of SPARC as a therapeutic target for medulloblastoma.nnCitation 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 2286.

Abstract 2291: Downregulation of uPA/uPAR inhibits epithelial to mesenchymal transition (EMT) in intermittent hypoxic DAOY and D283 medulloblastoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnMedulloblastoma is the most common, rapidly growing tumor of the cerebellum, which arises in childhood and has a tendency to metastasize via cerebrospinal fluid (CSF) pathways. In addition to surgical resection, post-operative radiotherapy and chemotherapy are the mainstays in treating patients with medulloblastoma. However, these tumors remain among the most challenging pediatric brain tumors. Factors related to the tumor microenvironment are being recognized as fundamental to tumor progression, increased resistance and metastasis. Hypoxia, one of these factors, leads to epigenetic and genetic adaptation of clones and increased EMT, invasiveness and metastasis. Thus, hypoxia is closely associated with tumorigenesis and provides tumors the ability to outgrow the existing vasculature. In this study, we show that intermittent hypoxia has a more pronounced effect than chronic hypoxia and is responsible for EMT, invasion, migration, and survival of hypoxic-conditioned medulloblastoma cells. We also show that Akt and SNAIL are activated under hypoxia, and this response is blocked by plasmid vector-mediated delivery of uPA/uPAR siRNA. The linkage of uPAR to Akt activation under hypoxia provides one mechanism by which uPAR may regulate SNAIL, and thus, promote EMT. The results presented here suggest that, in cells that express high levels of uPAR, induction and maintenance of EMT depends on simultaneous activation of numerous cell-signaling factors downstream of uPAR. This study provides valuable evidence of the anti-tumor efficacy of plasmid vector-mediated delivery of uPA/uPAR siRNA in hypoxic-conditioned medulloblastoma cells to target intermittent hypoxia-induced cell invasion, migration, angiogenesis and EMT and to achieve better therapeutic outcomes in the treatment of malignant medulloblastomasnnCitation 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 2291.

Abstract 511: Integrin-mediated inhibition of adhesion, migration and invasion in glioma xenografts by MMP-9, uPAR and cathepsin B gene silencing

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnInvolvement of MMP-9, cathepsin B and uPAR in glioma adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well studied and reported. In the present study, we have downregulated MMP-9, uPAR and cathepsin B genes by using siRNA plasmid constructs in glioma xenografts (4910 and 5310) to study their effects on adhesion, migration and invasion. We hypothesized that downregulation of these genes in combination will have additive/synergistic effect in inhibiting adhesion, migration and invasion of glioma xenografts and hence, treated these xenografts with MMP-9-uPAR and MMP-9-cathepsin B bicistronic plasmid constructs. MMP-9 (pM), uPAR (pU) and cathepsin B (pC) downregulation in glioma xenografts significantly inhibited their adhesion to several extracellular matrix proteins, such as collagen I (pM-39%, pU-51%, pC-57% inhibition in 4910 cells and pM-40%, pU-44%, pC-59% inhibition in 5310 cells), vitronectin (pM-51%, pU-45%, pC-57% inhibition in 4910 cells and pM-56%, pU-58%, pC-58% inhibition in 5310 cells), fibronectin (pM-36%, pU-48%, pC-48% inhibition in 4910 cells and pM-30%, pU-37%, pC-41% inhibition in 5310 cells), and laminin (pM-50%, pU-34%, pC-45% inhibition in 4910 cells and pM-48%, pU-52%, pC-65% inhibition in 5310 cells). Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B siRNA treatments as assessed by spheroid migration, migration through 8 μm transwell inserts, wound healing and matrigel invasion assays. As expected, downregulation of MMP-9 in combination either with uPAR (pMU) and cathepsin B (pMC) further inhibited the adhesion, migration and invasion of 4910 and 5310 glioma xenografts as compared to individual treatments. Furthermore, MMP-9, uPAR and cathepsin B siRNA treatments of these xenografts led to the downregulation of several alpha and beta integrins (such as α6, α9, αV, β1 and β3) as evidenced by reverse transcriptase PCR analysis. Immunofluorescence analysis revealed the reduced expression of these integrins in the glioma xenografts when treated with MMP-9, uPAR and cathepsin B siRNA either alone or in combination. Based on these findings, we conclude that downregulation of MMP-9, uPAR and cathespin B using siRNA plasmid constructs inhibits adhesion, migration and invasion of human glioma xenografts via the downregulation of integrins. Considering the recent clinical failures with integrin inhibitors and the existence of integrin inhibitor resistant cancer cells, the present study provides a novel and effective approach for cancer treatment by inhibiting the integrins while downregulating MMP-9, cathepsin B and uPAR.nnCitation 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 511.

Abstract 1322: Sensitizing radiation-induced integrin β-1 signaling by targeting uPAR in medulloblastoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnAlthough radiation is an effective therapy, it has several adverse effects, including the induction of secondary (new) tumor within the irradiated field. Several studies have reported this effect with medulloblastoma despite effective radiotherapy during the initial stages. Here, we attempt to capitalize on the radiation-induced aggressive behavior of medulloblastoma cells by examining migration, cell adhesion and matrigel invasion properties. Comparison at the mRNA levels between the matrigel-invading and non-invading cells (with and without radiation) clearly demonstrates the aggressiveness of irradiated medulloblastoma cells. RT-PCR analysis confirmed the increased expression of uPA, uPAR, focal adhesion kinase (FAK), N-Cadherin, and integrins like α3, α5, β1 and β6 in irradiated cells. Increased expression of uPAR and integrin β-1 in irradiated cells was also confirmed by immunofluorescence and immunoprecipitation assays. The interaction between uPAR and integrin β-1 and subsequent downstream signaling is a key process in malignant progression and metastasis and has been recently reported in mediating resistance to radiation. With this view, we aimed to study the effect of siRNA-mediated knockdown of uPAR on cell migration and adhesion in irradiated and non-irradiated medulloblastoma cells. Downregulation of uPAR not only reduced cell migration and adhesion, but also reduced the increased migration and adhesiveness associated with irradiated cells. Apart from specific knockdown of uPAR in siRNA-transfected cells, the increased expression of uPAR and integrin β-1 upon radiation were also reduced both at the transcript and protein levels. Studies of the uPAR/integrin β-1 downstream signaling cascade have shown that uPAR knockdown reduces both the expression and phosphorylation of FAK, paxillin, p130Cas and Rac-1, which are known to be actively involved in coordinating tyrosine kinase based signaling related to cell migration and adhesion. Our in vitro results demonstrate that targeting uPAR might sensitize the radiation-induced aggressiveness of medulloblastoma cells by affecting the uPAR/integrin β-1 signaling pathway.nnCitation 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 1322.