Kiranmai Alapati

Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

BACKGROUND Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes. METHODOLOGY/PRINCIPAL FINDINGS In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results. CONCLUSIONS/SIGNIFICANCE In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.

Cathepsin B and uPAR knockdown inhibits tumor-induced angiogenesis by modulating VEGF expression in glioma

Angiogenesis, which is the process of sprouting of new blood vessels from pre-existing vessels, is vital for tumor progression. Proteolytic remodeling of extracellular matrix is a key event in vessel sprouting during angiogenesis. Urokinase type plasminogen activator receptor (uPAR) and cathepsin B are both known to be overexpressed and implicated in tumor angiogenesis. In the present study, we observed that knockdown of uPAR and cathepsin B using puPAR (pU), pCathepsin B (pC), and a bicistronic construct of uPAR and cathepsin B (pCU) caused significant inhibition of angiogenesis by disrupting the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway-dependent expression of vascular endothelial growth factor (VEGF). Further, transcriptional suppression of uPAR and cathepsin B inhibited tumor-induced migration, proliferation of endothelial cells and decreased tumor-promoted expression of VEGF receptor-2, Rac1, gp91phox, cyclin D1, cyclin dependent kinase 4 and p-Rb in human dermal microvascular endothelial cell. Furthermore, U251 and SNB19 xenograft tissue sections from nude mice treated with pCU showed reduced expression of VEGF and CD31, which is a blood vessel visualization marker. Overall, results revealed that knockdown of uPAR and cathepsin B inhibited tumor-induced angiogenesis by disrupting the JAK/STAT pathway-dependent expression of VEGF. These data provide new insight in characterizing the pathways involved in the angiogenic cascade and for the identification of novel target proteins for use in therapeutic intervention for gliomas.

Co-depletion of cathepsin B and uPAR induces G0/G1 arrest in glioma via FOXO3a mediated p27Kip1 upregulation

Background Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined. Methodology/Principal Findings Cathespin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27Kip1 and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by αVβ3/PI3K/AKT/FOXO pathway as observed by the decreased αVβ3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27Kip1 and FOXO3a when treated with Ly294002 (10 µM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27Kip1 was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly. Conclusion/Significance Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27Kip1 accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.

Cathepsin B and uPAR regulate self-renewal of glioma-initiating cells through GLI-regulated Sox2 and Bmi1 expression

Cancer-initiating cells comprise a heterogeneous population of undifferentiated cells with the capacity for self-renewal and high proliferative potential. We investigated the role of uPAR and cathepsin B in the maintenance of stem cell nature in glioma-initiating cells (GICs). Simultaneous knockdown of uPAR and cathepsin B significantly reduced the expression of CD133, Nestin, Sox2 and Bmi1 at the protein level and GLI1 and GLI2 at the messenger RNA level. Also, knockdown of uPAR and cathepsin B resulted in a reduction in the number of GICs as well as sphere size. These changes are mediated by Sox2 and Bmi1, downstream of hedgehog signaling. Addition of cyclopamine reduced the expression of Sox2 and Bmi1 along with GLI1 and GLI2 expression, induced differentiation and reduced subsphere formation of GICs thereby indicating that hedgehog signaling acts upstream of Sox2 and Bmi1. Further confirmation was obtained from increased luciferase expression under the control of a GLI-bound Sox2 and Bmi1 luciferase promoter. Simultaneous knockdown of uPAR and cathepsin B also reduced the expression of Nestin Sox2 and Bmi1 in vivo. Thus, our study highlights the importance of uPAR and cathepsin B in the regulation of malignant stem cell self-renewal through hedgehog components, Bmi1 and Sox2.

Cord Blood Stem Cells Inhibit Epidermal Growth Factor Receptor Translocation to Mitochondria in Glioblastoma

Background 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. Methodology/Principle Findings 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 proteins in glioma cells both in vitro and in vivo. Conclusions/Significance 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.

uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton of glioma-initiating cells

Despite advances in radiotherapeutic and chemotherapeutic techniques and aggressive surgical resection, the prognosis of glioblastoma patients is dismal. Accumulation of evidence indicates that some cancer cells survive even the most aggressive treatments, and these surviving cells, which are resistant to therapy and are perhaps essential for the malignancy, may be cancer stem cells. The CD133 surface marker is commonly used to isolate these extremely resistant glioma-initiating cells (GICs). In the present study, GICs which tested positive for the CD133 marker (CD133+) were isolated from both the established U251 cell line and the 5310 xenograft glioma cell line to study the events related to the molecular pathogenesis of these cells. Simultaneous down-regulation of uPAR and cathepsin B by shRNA (pUC) treatment caused the disruption of radiation-induced complex formation of pPKC θ/δ, integrin β1 and PKC ζ, integrin β1 in glioma cells. Further, pUC treatment inhibited PKC/integrin signaling via FAK by causing disassociation of FAK and the cytoskeletal molecules vinculin and α-actinin. Also, we observed the inhibition of ERK phosphorylation. This inhibition was mediated by pUC and directed a negative feedback mechanism over the FAK signaling molecules, which led to an extensive reduction in the signal for cytoskeletal organization generating migratory arrest. Altogether, it can be hypothesized that knockdown of uPAR and cathepsin B using shRNA is an effective strategy for controlling highly invasive glioma cells and extremely resistant glioma-initiating cells.

uPAR and cathepsin B-mediated compartmentalization of JNK regulates the migration of glioma-initiating cells ☆

In the present study, we investigated the effect of simultaneous downregulation of uPAR and cathepsin B (pUC), alone or in combination with radiation, on JNK–MAPK signaling pathway in regulating the migration of non-GICs (glioma-initiating cells) and GICs. The increase in the expression of p-JNK with pUC treatment was mostly localized to nucleus whereas increase in the expression of p-JNK with radiation and overexpression of uPAR and cathepsin B was confined to cytoplasm of the cells. Depletion of cytosolic p-JNK with pUC treatment inhibited migration by downregulating the expression of the adapter proteins of the focal adhesion complex. We also observed that knockdown of uPAR and cathepsin B regulated the Ras–Pak-1 pathway to induce the translocation of p-JNK from cytosol to nucleus. In control cells, Pak-1 served as a functional inhibitor for MEKK-1, which inhibits the complex formation of MEKK-1 and p-JNK and thus inhibits the translocation of this complex into nucleus. Hence, we conclude that glioma cells utilize the availability of cytosolic p-JNK in driving the cells towards migration. Finally, treating the cells with pUC alone or in combination with radiation induced the translocation of the MEKK-1-p-JNK complex from cytosol to nucleus, thereby inhibiting the migration of glioma cells.

Mechanism of p27 Upregulation Induced by Downregulation of Cathepsin B and uPAR in Glioma

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. p27Kip1 (p27) is one of the major cyclin‐CDK regulators in the G1 phase. uPAR and cathepsin B downregulation was recently shown to induce p27 expression through PI3K/Akt/FOXO3a signaling. Since uPAR and cathepsin B knockdown also decreased phosphorylation of ERK, we hypothesized that ERK also has a role to play in p27 induction. As induction of p27 is due to an increase in gene transcription, we investigated the roles of c‐Myc and E2F1 transcription factors which have been shown to potently affect p27 promoter activity. In the present study, shRNA against cathepsin B and uPAR as well as specific inhibitors, Wortmannin (10 μM) and U0126 (10 μM), were used to determine the roles of AKT and ERK signaling on p27 expression. Immunoblot analysis demonstrated that downregulation of both p‐ERK and p‐AKT downstream of EGFR and β1 integrin are involved in the p27 upregulation. Cathepsin B and uPAR downregulation induced E2F1 and decreased phosphorylaion of pocket proteins and c‐Myc expression. CHIP analysis and luciferase expression studies confirmed the functional association of transcription factor E2F1 to the p27 promoter. Further, c‐Myc–Max interaction inhibitor studies showed an inverse pattern of c‐Myc and p27 expression. Also, cathepsin B and uPAR downregulation reduced tumor growth and increased p27 nuclear expression in vivo. In summary, cathepsin B and uPAR downregulation reduced p‐ERK levels and c‐Myc expression, increased expression of E2F1 and FOXO3a, decreased phosphorylation of pocket proteins and thus upregulated p27 expression in glioma cells.

Abstract 2617: uPAR and cathepsin B knockdown-induced nuclear translocation of JNK inhibits migration and induces apoptosis in glioma-initiating cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Treatment for glioblastoma (GBM) remains essentially palliative due to the aggressive infiltration of GBM cancer cells into normal brain tissue. In addition, the existence of a small subpopulation of highly resistant cells known as glioma-initiating cells (GICs), which escape radiation and chemotherapy-induced cell death, makes GBM currently incurable. Proteases, such as uPAR and cathepsin B that are responsible for cancer invasion and metastasis, are often detected in higher amounts in malignant tumors. In the present study, shRNA-mediated knockdown of uPAR and cathepsin B (pUC), alone or in combination with radiation, simultaneously inhibited migration and induced apoptosis of 5310 and 4910 non-GICs and GICs by regulating the JNK-MAPK pathway. Immunoblot and immunocytochemical analyses showed pUC treatment resulted in an increase in the levels of the phospho-JNK (p-JNK), which was mostly localized to the nucleus. We also observed an increase in the levels of p-JNK with radiation and with full-length uPAR and cathepsin B; however, it was confined to the cytoplasm of the cells. Depletion of cytosolic p-JNK with pUC treatment and/or with the JNK inhibitor decreased migration by downregulating the expression of the migratory molecules. Immunoprecipitation analysis and co-localization studies further confirmed the involvement of the cytosolic p-JNK in promoting migration of the glioma cells. The increase in the expression of nuclear p-JNK with pUC treatment increased the expression of apoptotic molecules, as observed by the western blot analysis. MTT assay revealed that non-GICs and GICs were rescued from pUC-induced cell death when treated in combination with a JNK inhibitor, which further confirms the importance of nuclear JNK in eliciting an apoptotic signal. In summary, cytosolic p-JNK aids cell migration while nuclear p-JNK drives cells toward cell death. As such, pUC treatment induced the translocation of p-JNK from cytoplasm to the nucleus, thereby simultaneously inhibiting migration and inducing apoptosis of non-GICs and GICs. Citation Format: Kiranmai Alapati, Divya Kesanakurti, Jasti S. Rao. uPAR and cathepsin B knockdown-induced nuclear translocation of JNK inhibits migration and induces apoptosis in glioma-initiating cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2617. doi:10.1158/1538-7445.AM2013-2617

Abstract 475: uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton and migration of glioma initiating cells

Glioblastoma (GBM) remains one of the most fatal and least successfully treated solid tumors. It is evident that some cancer cells with stem cell properties (known as glioma initiating cells; GICs) survive conventional treatments and are involved in the recurrence of tumors. In the present study, CD133+ GICs were isolated from established U251 and 5310 glioma cell lines and subjected to radiation and transfection with shRNA against uPAR and cathepsin B so that we could study the events related to cell migration. Simultaneous downregulation of uPAR and cathepsin B (pUC) caused the disruption of radiation-induced complex formation of pPKC∈/α:integrinβ1 and PKC∈:integrinβ1 in glioma cells as well as in GICs as was observed by immunoprecipitation analysis. Further, pUC treatment also inhibited the PKC/integrin signal through FAK by disintegrating the association of FAK and the cytoskeletal molecules vinculin and α-actinin. It was also observed that inhibition of radiation-induced ERK phosphorylation by pUC directed a negative feedback mechanism over the FAK signaling molecules that led to an extensive reduction in the cytoskeletal organization, and thereby generating a migratory arrest. Further confirmation was obtained by treating the cells with U0126 (10 µM), a MEK inhibitor, and rottlerin (200 µM), a PKC inhibitor. pUC treatment efficiently inhibited the co-localization of pPKC∈/α/integrinβ1, PKC∈/integrinβ1, FAK/vinculin and FAK/α-actinin in the brain tumor sections of mice implanted with glioma cells and GICs. In conclusion, shRNA-mediated knockdown of uPAR and cathepsin B could be an effective treatment strategy for controlling highly invasive glioma cells and treatment-resistant glioma initiating 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 475. doi:1538-7445.AM2012-475