Christopher S. Gondi

Abstract 136: Suppression of the uPA/uPAR system causes the overexpression of miR124 and suppresses stemness in glioma stem cells

The invasiveness and destructiveness of malignant neoplasms in the central nervous system are of great clinical importance. Higher-grade tumors, such as glioblastomas, are associated with poor prognosis, and patients’ average survival is only 8 to 12 months after chemotherapy and/or radiotherapy. This poor prognosis reflects the resistance of tumor cells to radiation and cytotoxic agents as well as the difficulty in achieving total tumor resection. Highly infiltrative gliomas are known to overexpress both uPA and uPAR. MicroRNAs (miRNAs) are essential post-transcriptional regulators known to determine cell identity and fate and are also known to be involved not only in development and differentiation but also in glioma progression. Acquisition of EMT has also recently been linked to stem cell phenotypes which is mediated by microRNAs. However, the molecular mechanism underlying EMT regulation still remains elusive. In the present study, we used glioma initiating cells (GICs), which show stem cell-like character, and determined whether their stemness can be suppressed by targeting the uPA/uPAR system. We raised glioma GICs from U87MG and 4910 glioma xenograft cells and observed that GICs expressed 2- to 3-fold increased levels of uPA and uPAR when compared to non-GIC. Further, the simultaneous downregulation of both uPA and uPAR suppressed GIC from establishing intracranial tumors in nude mice. Further, to understand whether this suppression of GIC involved microRNAs, we profiled miR expression in U87 cells and observed that miR124 and miR200a were significantly overexpressed. Using miRanda analysis, we determined that miR124 targets the regulation of Lhx-2 and Lhx-2 is over expressed (3- to 4-fold) in GIC when compared to non-GIC. To determine whether this suppression of Lhx-2 by downregulation of uPA and uPAR is mediated via miR124, we overexpressed miR124 in U87MG and 4910 glioma xenograft GICs and observed that miR124-overexpressed cells failed to establish intracranial tumors in nude mice. Our results demonstrate that the uPA/uPAR system may be involved in GIC maintenance via suppression of miR124. 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 136. doi:1538-7445.AM2012-136

Abstract 5212: Irradiation-induced uPAR promotes stemness via Wnt/β-catenin signaling in medulloblastoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Urokinase plasminogen activator receptor (uPAR) is known to promote invasion, migration and metastasis in cancer cells. In this study, we show that ionizing radiation (IR) induced uPAR has a role in Wnt/β-catenin signaling and mediates induction of cancer stem cells (CSC)-like properties in medulloblastoma cell lines UW228 and D283. We observed that IR induced the expression of uPAR, CSC markers such as CD44 and Msi-1, and activates Wnt/β-catenin signaling molecules. Overexpression of uPAR after IR treatment led to the activation of Wnt signaling, which was demonstrated by an increase in nuclear translocation of β-catenin and β-catenin-Lef/Tcf-mediated transactivation, thereby promoting cancer stemness. Quercetin, a potent Wnt/β-catenin inhibitor suppressed uPAR and uPAR-mediated Wnt/β-catenin activation. Treatment with shRNA specific for uPAR (pU) suppressed the β-catenin-Tcf/Lef-mediated transactivation both in vitro and in vivo. Further, we show that uPAR is physically associated with the Wnt effector molecule β-catenin using immunocytochemistry and immunohistochemistry; these results were confirmed by immunoprecipitation analysis. Most interestingly, we demonstrate for the first time that the localization of uPAR in the nucleus is associated with transcription factors (TF) and their specific response elements. The association of uPAR with β-catenin-Tcf/Lef complex and various other TF involved in neurogenesis during embryonic development and cancer demonstrates the receptors possible role at generating CSC-like properties in primitive neuroectodermal tumor (PNET) cells of medulloblastoma. Considering all of the data, we conclude that uPAR is a potent activator of stemness, and the targeting of uPAR in combination with radiation has significant therapeutic implications. 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 5212. doi:1538-7445.AM2012-5212

Abstract 4358: Notch signaling regulates tumor-induced angiogenesis in SPARC-overexpressed neuroblastoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Despite existing aggressive treatment modalities, survival for advanced stage neuroblastoma remains poor with significant long-term illness in disease survivors. Advance stage disease features are associated with tumor vascularity, and as such, angiogenesis inhibitors may prove useful along with current therapies. SPARC (also known as BM-40 or osteonectin), a matricellular protein, is known to inhibit the proliferation and migration of endothelial cells stimulated by growth factors and induces endothelial cell apoptosis. Here, we sought to determine the effect of SPARC on neuroblastoma tumor cell-induced angiogenesis and to decipher the molecular mechanisms involved in angiogenesis inhibition. SPARC overexpression in neuroblastoma cells inhibited neo-vascularization in vivo as determined by the mouse dorsal air sac model. In addition, conditioned medium from SPARC-overexpressed neuroblastoma cancer cells (pSPARC-CM) inhibited endothelial tube formation in vitro. SPARC-overexpressed tumor cell conditioned medium decreased proliferation (as determined by MTT assay and Ki-67 immunofluorescence) and induced apoptosis in endothelial cells (as determined by TUNEL assay). Furthermore, SPARC-overexpressed tumor cell conditioned medium inhibited expression of the pro-angiogenic molecules VEGF, FGF, PDFG, and MMP-9 as determined by human angiogenesis-PCR array. In addition to the inhibition of growth factors, SPARC-overexpressed tumor cell conditioned medium inhibited Notch signaling molecules in HMEC cells as determined by Western blotting. Conditioned medium from neuroblastoma cells overexpressed with Notch intracellular domain after SPARC overexpression, induced growth factors and led to increased tube formation in vitro. In vivo endothelial cell death was confirmed by co-localization studies with TUNEL assay, and an endothelial marker, CD31, in tumor sections from SPARC-overexpressed mice. Our data collectively suggest that SPARC overexpression induces endothelial cell apoptosis in vivo and inhibits angiogenesis both in vitro and in vivo. 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 4358. doi:1538-7445.AM2012-4358

Abstract 1294: Suppression of uPA in chemo-sensitization and p53 regulation in pancreatic cancer cells

Despite new knowledge of the molecular profile of pancreatic cancer and its precursor lesions, survival rates have changed very little over the last 40 years. The ability to predict which patients would benefit most from surgical intervention and/or chemotherapy would be a great clinical asset. Researchers have demonstrated that uPA is frequently present in a higher concentration in the serum of pancreaticobiliary cancer patients. It is known that uPA is involved in ECM degradation and has been correlated with malignant transformation of cancer cells. Emerging data have suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor called cancer stem cells (CSCs) or cancer initiating cells. Under in vitro cell culture conditions, these cells possess sphere-forming ability, which is known to be one of the properties of CSCs. Interestingly, the significance of this sphere formation ability still remains unclear. Recent studies have demonstrated that these sphere-forming cells acquired chemoresistance to cisplatin. In the present study, as observed by FACS analysis, we show that a sub-population of MIA PaCa-2 and PANC-1 pancreatic cancer cells possess cancer stem cell properties. RNAi-mediated suppression of uPA in these cells retarded their tumor forming ability in a nude mouse model. We also observed that uPA-suppressed MIA PaCa-2 and PANC-1 cells, after supplementation with uPA protein regained their cancer stem cell phenotype and showed increased expression of Lhx-2. To determine whether these sub-population cells possess any chemoresistance, we treated these cells with varying concentrations of gemcitabine (0-1000 nM) and observed that these cells were resistant to gemcitabine even at the 1000 nM concentration. Further suppression of uPA using siRNA in these cells sensitized these cells to gemcitabine. In addition, to determine whether uPA has any transcriptional role, we examined whether uPA interacted with any known transcription factors we used a TF-protein array. We observed that uPA interacted with Hoxa-5 very strongly. To further determine whether the interaction of uPA with Hoxa-5 invokes any regulatory response, we used a p53 promoter known to possess Hoxa-5 binding regions to drive a Luciferace gene, both with and without the presence of uPA. We observed that uPA retarded p53 promoter activity. Finally, nude mice implanted with MIA PaCa-2 cancer stem cells were susceptible to gemcitabine treatment after uPA downregulation. Taken together, these results indicate that targeting uPA can sensitize chemoresistant pancreatic cells to gemcitabine. 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 1294. doi:1538-7445.AM2012-1294

Abstract 3465: shRNA against uPA and uPAR enhances secretion of sVEGFr1 in tumor endothelial and glioblastoma cells independent of GMCSF

The uPA/uPAR system is known to play a critical role in angiogenesis of glioblastomas. Previously, using protein array antibody analysis, we demonstrated that shRNA against uPA and uPAR (pU2) inhibits angiogenesis in vitro and in vivo by regulating a number of pro-angiogenic, angiostatic and anti-angiogenic molecules. GMCSF, a pleiotropic cytokine that stimulates proliferation, differentiation, and survival of various hematopoietic cells, was significantly inhibited in U87MG and 4910 cells co-cultured with endothelial cells and transfected with pU2. In this study, we evaluated the effect of shRNA against uPA and uPAR on GMCSF, sVEGFr1, GMCSF receptors and GMCSF-induced signaling and its significance on angiogenesis in glioblastoma and endothelial cells. ELISA revealed that simultaneous knockdown of uPA and uPAR in U87MG, 4910 and HMEC cells decreased secretion of GMCSF but enhanced the secretion of sVEGFr1, a known scavenger of VEGF-A. The results of the western blot analysis were consistent with our ELISA results. In endothelial cells treated with shRNA against GMCSF, recombinant uPA (ruPA) resulted in activation of GMCSFR-α and GMCSFR-β at very early time points. Moreover, endothelial tube formation was inhibited with the addition of rsVEGFr1, and antibody to rsVEGFr1 blocked GMCSF-induced tube formation in endothelial cells. The addition of ruPA to endothelial and glioblastoma cells activated phosphorylation of JAK2 and STAT3. p27, an effector of the GMCSF signaling pathway, was downregulated in puPA-, puPAR- and pU2-transfected U87MG and 4910 cells. To determine the significance of these events in vivo, nude mice were checked for levels of GMCSF and sVEGFr1 before and after tumor formation and also after treatment with puPA, puPAR and pU2. Results showed increased levels of sVEGFr1 but decreased levels of GMCSF in shRNA treated mice in comparison to controls. Taken together, these results suggest that knockdown of uPA/uPAR in tandem induces sVEGFr1, which plays a vital role in inhibition of angiogenesis, and its secretion is independent of GMCSF in endothelial cells. 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 3465. doi:10.1158/1538-7445.AM2011-3465

Abstract 3319: Cell cycle regulation of glioblastoma progression by cord blood stem cells is mediated by downregulation of cyclin D1

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, is considered a rate-limiting step during cell cycle progression through the G1 phase. In the present study, human umbilical cord blood stem cells (hUCBSC) co-cultured with glioblastoma cells (U251 and 5310) not only induced G0-G1 phase arrest, but also reduced the number of cells at the S and G2-M phases of the cell cycle. Western and FACS analyses of cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated decreased expression of cyclin D1 in the presence of hUCBSC. Treatment with hUCBSC decreased expression of the cyclin D1 and CDK4 complex as well as cyclin D1 and CDK6 complexes. However, there was no significant change in the CDK4 and CDK6 complex. Our results indicate that hUCBSC can attenuate uncontrolled cell cycle progression of glioma cells by downregulating the expression levels of cyclin D1 and its partner kinases CDK4 and CDK6 at the cell cycle level. Our data elucidate a model to regulate glioma cell cycle progression in which hUCBSC act to control cyclin D1 induction and its partner kinases, CDK4 and CDK6, by mediating cell cycle arrest via G0-G1 arrest. 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 3319. doi:10.1158/1538-7445.AM2011-3319

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 1529: Knockdown of cathepsin B and uPAR inhibits CD151 and α3β1 integrin-mediated cell adhesion in glioma

Glioma is a highly complex brain tumor characterized by deregulation of proteins and genes that play important roles in tumor metastasis. CD151, a member of the tetraspanin family of proteins, tightly associates with integrins and accelerates cell adhesion as a modulator of actin cytoskeletal reorganization. Cathepsin B and uPAR are both overexpressed in gliomas and postulated to play central roles in the mediation of glioma metastasis. In the present study, cathepsin B and uPAR were downregulated in U251 and 4910 glioma cells using siRNA plasmid constructs. We evaluated the involvement of CD151 and its associated signaling molecules in adhesion potential. Immunoblot analysis revealed efficient downregulation of cathepsin B and uPAR after siRNA treatments. In addition, siRNA treatments significantly inhibited glioma cell adhesion to laminin as compared to other extracellular matrix proteins. Brain glioma tissue array analysis revealed the expression of CD151 in several tumor samples when compared with normal brain tissue. Further, treatment of glioma cells with cathepsin B and uPAR siRNA led to the downregulation of CD151 and laminin-binding integrins, α3 and β1. Co-localization and immunoprecipitation experiments revealed that downregulation of cathepsin B and uPAR decreased the interaction of CD151 with uPAR/cathepsin B. Transcriptional suppression of CD151 and α3 and β1 integrins using siRNA significantly decreased glioma cell adhesion to laminin. In addition, downregulation of cathepsin B, uPAR, CD151, α3 and β1 significantly decreased the expression of N-cadherin and β-catenin. Studies on the downstream signaling cascade of uPAR/CD151 have shown that phosphorylation of SRC, FAK and Paxillin were reduced with knockdown of cathepsin B, uPAR and CD151. Treatment with the bicistronic construct reduced CD151, α3β1 integrin, N-cadherin and β-catenin expression levels in pre-established glioma tumors in nude mice as well as in tissue lysates. In conclusion, our results show that downregulation of cathespin B and uPAR alone and in combination inhibit glioma cell adhesion by downregulating CD151 and its associated signaling molecules in vitro and in vivo. This study may be useful in dissecting the pathways involved in tumor cell adhesion and for the identification of novel target proteins for therapeutic intervention of gliomas. 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 1529. doi:10.1158/1538-7445.AM2011-1529

Abstract 3393: Extrinsic uPA initiates glioma stem cell transformation under hypoxic conditions via Lhx-2

Emerging evidence indicate that the capacity of a tumor to grow and propagate resides primarily in a small population of tumor cells, which are known as cancer stem cells. The origin of these cancer stem cells remains controversial. Hypoxia promotes tumor progression through multiple mechanisms, including modification of angiogenesis, metabolic switches and invasion. Researchers have demonstrated that hypoxia is one of the factors necessary for neural stem cell tropism to glioma. Although hypoxia has been shown to maintain multiple normal stem cell populations, its role in cancer stem cells is fairly unknown. Recently, researchers have identified hypoxia as a critical microenvironmental factor in regulating self-renewal of cancer stem cells, which is partially mediated by enhancing the activity of stem cell factors such as Oct4, c-Myc and Nanog. In this study, we show that under hypoxic conditions, urokinase plasminogen activator (uPA) is one of the factors necessary for the maintenance and transformation of glioma cells to glioma stem cells. Glioma cells grown under hypoxic conditions showed an increase in expression of stem cell markers Oct-4, c-Myc, CD133 and Stro-1. Supplementing normoxic or hypoxic cells with external uPA increased Lhx-2 expression and was not influenced by the presence or absence of native uPA. Hypoxic cells down regulated for Lhx-2 did not show activation of stem cell marker proteins Oct-4, c-Myc, CD133 and Stro-1. Hypoxic cells subcutaneously implanted into nude mice developed at least 2-fold larger tumors when compared to normoxic cells. Intratumoral injection of plasmids expressing siRNA targeting uPA retarded tumor development in both normoxic and hypoxic tumors. In contrast, injection of plasmids expressing siRNA targeting Lhx-2 retarded hypoxic tumors with reduced effect on normoxic tumors. In conclusion, our results demonstrate that a hypoxic environment in a high uPA environment can induce glioma stem cell transformation mediated by Lhx-2 and suppression of uPA and Lhx-2 can specifically target glioma stem cell populations. 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 3393. doi:10.1158/1538-7445.AM2011-3393

Abstract 5369: AN019 in combination with ionizing radiation retards intracranial tumor growth and inhibits angiogenic induction in vitro and in vivo

Successful treatment of malignant glioma is hampered by the development of resistance to alkylating agents such as Temozolomide (TMZ). The addition of radiation to a TMZ treatment regime has shown promising results with reported survivability of up to 5 years. One of the side effects of Temozolomide is the increased activation of angiogenic factors which may work contrary to the desired result. In this report we demonstrate the development of a novel compound AN019, showing specific VEGF suppression accompanied with anti-angiogenic and anti-proliferative properties in gliomas. We used U87, a glioma cell line, and EGFRvIII over-expressing glioma xenograft 4910 cells for this study. Deduced IC50 values indicated that AN019 had greater anti-proliferative effect than Temozolomide (U87: AN019=103µM, TMZ=768µM; 4910: AN019=169µM, TMZ=582µM). Matrigel invasion assay indicated that AN019 retarded the invasive potential of both U87 and 4910 glioma cells over TMZ treated cells which was enhanced after radiation treatment. In-vitro angiogenic and migration assays showed an almost complete inhibition of angiogenic induction and migration in U87 and 4910 cells after AN019 treatment when compared to TMZ. Cell cycle analysis revealed that 100µg/ml concentration of AN019 was sufficient to achieve >70% apoptosis in both U87 and 4910 cells, whereas 150µg/ml of TMZ was required to achieve the same response. Western blot and ELISA for the expression of VEGF levels indicated that lower concentrations of AN019 (U87=86±3µg/ml and 12±1µg/ml with radiation; 4910=100±1µg/ml and 6µg/ml with radiation) were required for 50% inhibition of VEGF expression. TMZ treatment required a higher concentration to achieve similar 50% inhibition of VEGF expression (U87=80±10µg/ml and 12±3µg/ml with radiation; 4910=100±3µg/ml and 27±3µg/ml with radiation). Animal studies showed that oral doses of AN019 (20mg/kg) regressed tumors in all animals and 70% of the animals showed complete absence of intracranial tumors, whereas TMZ (100mg/kg) treated animals also showed regression of tumors with a 30% cure rate. Overall, our results demonstrate that AN019 has anti-angiogenic, anti-proliferative and anti-tumorogenic properties which may have clinical implications. 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 5369.