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.

Urokinase-type plasminogen activator receptor (uPAR)-mediated regulation of WNT/ß-catenin signaling is enhanced in irradiated medulloblastoma cells.

Urokinase plasminogen activator receptor (uPAR) is known to promote invasion, migration, and metastasis in cancer cells. In this report, we showed that ionizing radiation (IR)-induced uPAR has a role in WNT-ß-catenin signaling and mediates induction of cancer stem cell (CSC)-like properties in medulloblastoma cell lines UW228 and D283. We observed that IR induced the expression of uPAR and CSC markers, such as Musashi-1 and CD44, and activated WNT-7a-ß-catenin signaling molecules. Overexpression of uPAR alone or with IR treatment led to increased WNT-7a-ß-catenin-TCF/LEF-mediated transactivation, thereby promoting cancer stemness. In contrast, treatment with shRNA specific for uPAR (pU) suppressed WNT-7a-ß-catenin-TCF/LEF-mediated transactivation both in vitro and in vivo. Quercetin, a potent WNT/ß-catenin inhibitor, suppressed uPAR and uPAR-mediated WNT/ß-catenin activation, and furthermore, addition of recombinant human WNT-7a protein induced uPAR, indicating the existence of a mutual regulatory relationship between uPAR and WNT/ß-catenin signaling. We showed that uPAR was physically associated with the WNT effector molecule ß-catenin on the membrane, cytoplasm, and nucleus of IR-treated cells and CSC. Most interestingly, we demonstrated for the first time that localization of uPAR in the nucleus was associated with transcription factors (TF) and their specific response elements. We observed from uPAR-ChIP, TF protein, and protein/DNA array analyses that uPAR associates with activating enhancer-binding protein 2α (AP2a) and mediates ß-catenin gene transcription. Moreover, association of uPAR with the ß-catenin·TCF/LEF complex and various other TF involved during embryonic development and cancer indicates that uPAR is a potent activator of stemness, and targeting of uPAR in combination with radiation has significant therapeutic implications.

Notch signaling regulates tumor-induced angiogenesis in SPARC-overexpressed neuroblastoma.

Despite existing aggressive treatment modalities, the prognosis 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. The matricellular protein, secreted protein acidic and rich in cysteine (SPARC), is known to inhibit proliferation and migration of endothelial cells stimulated by growth factors. 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. Conditioned medium from SPARC-overexpressed neuroblastoma cells (pSPARC-CM) inhibited endothelial tube formation, cell proliferation, induced programmed cell death and suppressed expression of pro-angiogenic molecules such as VEGF, FGF, PDGF, and MMP-9 in endothelial cells. Further analyses revealed that pSPARC-CM-suppressed expression of growth factors was mediated by inhibition of the Notch signaling pathway, and cells cultured on conditioned medium from tumor cells that overexpress both Notch intracellular domain (NICD-CM) and SPARC resumed the pSPARC-CM-suppressed capillary tube formation and growth factor expression in vitro. Further, SPARC overexpression in neuroblastoma cells inhibited neo-vascularization in vivo in a mouse dorsal air sac model. Furthermore, SPARC overexpression-induced endothelial cell death was observed by co-localization studies with TUNEL assay and an endothelial marker, CD31, in xenograft tumor sections from SPARC-overexpressed mice. Our data collectively suggest that SPARC overexpression induces endothelial cell apoptosis and inhibits angiogenesis both in vitro and in vivo.

Suppression of tumor cell invasiveness and in vivo tumor growth by microRNA-874 in non-small cell lung cancer.

MicroRNAs are a novel family of small non-coding RNAs that regulate the expression of several genes involved in normal development as well as human disorders including cancer. Here we show that miR-874 plays a tumor suppressor role in non-small cell lung cancer (NSCLC) in vitro and in vivo. In silico target prediction analysis revealed numerous genes associated with tumor progression including MMP-2 and uPA as the putative target genes of miR-874. Our preliminary in situ hybridization experiments demonstrated the diminution of miR-874 expression in lung cancer tissues compared to their normal counter parts. Overexpression of miR-874 in CD133-positive cancer stem cell (CSC) population led to a significant loss in CSC-phenotype and enhanced sphere de-differentiation into epithelial-like cells. Restoration of miR-874 expression drastically reduced cell invading ability in comparison to mock and control-miR-treated cells by suppressing the protein levels of MMP-2 and uPA. In in vivo experiments, miR-874 treatment decreased orthotopic tumor growth in nude mice compared to mock and control-miR treatments. Further, the immunoreactivity of human anti-MMP-2 and anti-uPA was significantly reduced in tumor sections from mice that received miR-874 treatment. In conclusion, our study highlights the possible tumor suppressor role of miR-874 in NSCLC-initiating cells and suggests miR-874 as a potential target in the treatment of NSCLC.

Knockdown of cathepsin B and uPAR inhibits CD151 and α3ß1 integrin-mediated cell adhesion and invasion in glioma.

Glioma is a highly complex brain tumor characterized by the dysregulation of proteins and genes that leads to tumor metastasis. Cathepsin B and uPAR are overexpressed in gliomas and they are postulated to play central roles in glioma metastasis. In this study, efficient downregulation of cathepsin B and uPAR by siRNA treatments significantly reduced glioma cell adhesion to laminin as compared to vitronectin, fibronectin, or collagen I in U251 and 4910 glioma cell lines. Brain glioma tissue array analysis showed high expression of CD151 in clinical samples when compared with normal brain tissue. Cathepsin B and uPAR siRNA treatment led to the downregulation of CD151 and laminin-binding integrins α3 and ß1. Co-immunoprecipitation experiments revealed that downregulation of cathepsin B and uPAR decreased the interaction of CD151 with uPAR cathepsin B, and α3ß1 integrin. Studies on the downstream signaling cascade of uPAR/CD151/α3ß1 integrin have shown that phosphorylation of FAK, SRC, paxillin, and expression of adaptor cytoskeletal proteins talin and vinculin were reduced with knockdown of cathepsin B, uPAR, and CD151. Treatment with the bicistronic construct reduced interactions between uPAR and CD151 as well as lowering α3ß1 integrin, talin, and vinculin expression levels in pre-established glioma tumors of nude mice. In conclusion, our results show that downregulation of cathepsin B and uPAR alone and in combination inhibit glioma cell adhesion by downregulating CD151 and its associated signaling molecules in vitro and in vivo. Taken together, the results of the present study show that targeting the uPAR-cathepsin B system has possible therapeutic potential.

Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration.

BACKGROUND: Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9ß1 signaling. Our recent studies have clearly demonstrated the role of α9ß1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of α9ß1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. METHODS: MMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis. RESULTS: Immunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p130Cas were reduced with simultaneous knockdown of both MMP-9 and uPAR. CONCLUSIONS: Taken together, our results from the present and earlier studies clearly demonstrate that α9ß1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9ß1 integrin and iNOS levels.

Molecular mechanisms of glioma invasiveness: the role of proteases.

The invasive nature of brain-tumour cells makes an important contribution to the ineffectiveness of current treatment modalities, as the remaining tumour cells inevitably infiltrate the surrounding normal brain tissue, which leads to tumour recurrence. Such local invasion remains an important cause of mortality and underscores the need to understand in more detail the mechanisms of tumour invasiveness. Several proteases influence the malignant characteristics of gliomas--could their inhibition prove to be a useful therapeutic strategy?

uPAR and cathepsin B downregulation induces apoptosis by targeting calcineurin A to BAD via Bcl-2 in glioma.

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are postulated to play key roles in glioma invasion. Calcineurin is one of the key regulators of mitochondrial-dependent apoptosis, but its mechanism is poorly understood. Hence, we studied subcellular localization of calcineurin after transcriptional downregulation of uPAR and cathepsin B in glioma. In the present study, efficient downregulation of uPAR and cathepsin B increased the translocation of calcineurin A from the mitochondria to the cytosol, decreased pBAD (S136) expression and its interaction with 14-3-3ζ and increased the interaction of BAD with Bcl-xl. Co-depletion of uPAR and cathepsin B induced mitochondrial translocation of BAD, activation of caspase 3 as well as PARP and cytochrome c and SMAC release. These effects were inhibited by FK506 (10 µM), a specific inhibitor of calcineurin. Calcineurin A was co-localized and also co-immunoprecipitated with Bcl-2. This interaction decreased with co-depletion of uPAR and cathepsin B and also with Bcl-2 inhibitor, HA 14-1 (20 µg/ml). Altered localization and interaction of calcineurin A with Bcl-2 was also observed in vivo when uPAR and cathepsin B were downregulated. In conclusion, downregulation of uPAR and cathepsin B induced apoptosis by targeting calcineurin A to BAD via Bcl-2 in glioma.

N-cadherin mediates angiogenesis by regulating monocyte chemoattractant protein-1 expression via PI3K/Akt signaling in prostate cancer cells.

Over the past decade, evidence continues to mount showing that N-cadherin is a critical protein in cancer progression and metastasis. In the present study, we evaluated the expression of N-cadherin in human prostate cancer tissue specimens and cell lines. Enhanced expression of N-cadherin was observed in both the malignant and bone-metastasized prostate tissue specimens compared to the healthy prostate tissues. Consistent with the tissue array data, N-cadherin was highly expressed in PC3, but not in Du145 and LNCaP human prostate cell lines. Based on cell to cell binding assay, we found that N-cadherin expression facilitates homotypic interaction between human prostate cancer cells and human microvascular endothelial cells (HMEC). Human angiogenesis antibody array and in vitro angiogenesis assay showed that siRNA-mediated knockdown of N-cadherin reduced the secretion of monocyte chemoattractant protein-1 (MCP-1), which played a potential role in stimulating capillary network formation of HMEC. Additionally, culture supernatant of Du145 cells transfected with full-length N-cadherin expressing plasmid showed increased MCP-1 expression and chemoattractant ability compared to normal Du145 cells. Further, we noticed that blocking PI3K activity inhibited N-cadherin mediated MCP-1 expression. Our data demonstrated that N-cadherin in prostate cancer cell mediates cell-cell adhesion and regulates MCP-1 expression via the PI3K/Akt signaling pathway.

Specific knockdown of uPA/uPAR attenuates invasion in glioblastoma cells and xenografts by inhibition of cleavage and trafficking of Notch -1 receptor.

BACKGROUND: uPA/uPAR is a multifunctional system that is over expressed in many cancers and plays a critical role in glioblastoma (GBM) invasion. Previous studies from our lab have also shown that uPA/uPAR down regulation inhibits cancer cell invasion in SNB 19 GBM cells. METHODS: As Notch 1 is known to be over expressed and promotes invasion in glioblastoma, we therefore tested our hypothesis of whether down regulation of uPA/uPAR, singly or in tandem, attenuates GBM invasion via Notch 1 receptor. Targeted down regulation of uPA/uPAR, either singly or simultaneously, inhibited the anchorage independent growth of U251MG and GBM xenograft cell lines 4910 and 5310 as assessed by soft agar colony formation assay. Expression of all four Notch receptors was confirmed in GBM tissue array analysis by immunohistochemistry. RESULTS: Down regulation of uPA/uPAR, either singly or simultaneously, in U251 MG and tumor xenografts inhibited the cleavage of the Notch receptor between the Gly 1743 and Val 1744 positions, thereby suggesting inhibition of activated cytosolic fragment-related Notch gene transcription. Morphological analysis confirmed inhibition of NICD when U251 MG cells were treated with puPA, puPAR or pU2. uPA/uPAR down regulation inhibited Notch 1 mRNA in all three examined cell lines. uPA/uPAR shRNA down regulated nuclear activation of NF-κB subunits and phosphorylation of AKT/mTOR pathway in U251 MG and GBM xenografts. puPA down regulated NICD and HES induced phosphorylation of AKT/ERK and NF-κB. Down regulation of Notch 1 using siRNA inhibited uPA activity as shown by fibrinogen zymography. It also decreased uPA expression levels as shown by western blotting. Exogenous addition of uPA activated Notch 1 in uPAR antisense U251 MG cells and also in uPAR antisense cells transfected with siRNA against Delta and Jagged. The Notch 1 receptor co-localized with LAMP-1, a marker for lysosomes in uPA, uPAR and U2, down regulated U251 MG cells which probably indicates inhibition of Notch 1 receptor trafficking in GBM cells. Notch 1 expression was significantly inhibited in puPA- and pU2-treated pre-established intracranial tumors in mice. CONCLUSIONS: Overall our results show that down regulation of uPA/uPAR, either singly or simultaneously, could be an effective approach to attenuate Notch 1 receptor cleavage, signaling and endosomal trafficking in U251MG cells and xenografts, and ultimately inhibiting GBM invasion.

p53- and Bax-mediated apoptosis in injured rat spinal cord.

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. p53-mediated mitochondrial apoptosis is likely to be an important mechanism of cell death in spinal cord injury. However, the signaling cascades that are activated before DNA fragmentation have not yet been determined. DNA damage-induced, p53-activated neuronal cell death has already been identified in several neurodegenerative diseases. To determine DNA damage-induced, p53-mediated apoptosis in spinal cord injury, we performed RT-PCR microarray and analyzed 84 DNA damaging and apoptotic genes. Genes involved in DNA damage and apoptosis were upregulated whereas anti-apoptotic genes were downregulated in injured spinal cords. Western blot analysis showed the upregulation of DNA damage-inducing protein such as ATM, cell cycle checkpoint kinases, 8-hydroxy-2'-deoxyguanosine (8-OHdG), BRCA2 and H2AX in injured spinal cord tissues. Detection of phospho-H2AX in the nucleus and release of 8-OHdG in cytosol were demonstrated by immunohistochemistry. Expression of p53 was observed in the neurons, oligodendrocytes and astrocytes after spinal cord injury. Upregulation of phospho-p53, Bax and downregulation of Bcl2 were detected after spinal cord injury. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis taking place after spinal cord injury. In addition, we carried out immunohistochemical analysis to confirm Bax translocation into the mitochondria and activated p53 at Ser³9². Expression of APAF1, caspase 9 and caspase 3 activities confirmed the intrinsic apoptotic pathway after SCI. Activated p53 and Bax mitochondrial translocation were detected in injured spinal neurons. Taken together, the in vitro data strengthened the in vivo observations of DNA damage-induced p53-mediated mitochondrial apoptosis in the injured spinal cord.

[Retracted] Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

Following the publication of the above paper, we were contacted by the University of Illinois at Chicago, to request the retraction of the above article. Following a formal institutional investigation, the investigation panel concluded that the images in question had falsifying elements. Regarding the above study, the specific allegations that were investigated were that of falsifying elements of Fig. 6A, row 2, columns 2 and 3. Following a review of this paper conducted independently by the Editor of International Journal of Oncology, the Editor concurred with the conclusions of the investigation panel, and therefore the above paper has been retracted from the publication. We also tried to contact the authors, but did not receive a reply. The Editor apologizes to the readership for the inconvenience caused.[the original article was published in International Journal of Oncology 40: 509­518, 2012; DOI: 10.3892/ijo.2011.1255].

[Retracted] Oncogenic role of p53 is suppressed by si­RNA bicistronic construct of uPA, uPAR and cathepsin­B in meningiomas both in vitro and in vivo.

Following the publication of the above paper, we were contacted by the University of Illinois at Chicago, to request the retraction of the above article. Following a formal institutional investigation, the investigation panel concluded that the images in question had falsifying elements. Regarding the above study, the specific allegations that were investigated were that of falsifying elements of Fig. 1B, bottom panel, columns 2 and 3; Fig. 4A, top panel, columns 4, 5 and 6, and middle panel, columns 1, 2 and 3; and Fig. 7D, row 1, column 1 and row 2, column 1.
Following a review of this paper conducted independently by the Editor of International Journal of Oncology, the Editor concurred with the conclusions of the investigation panel, and therefore the above paper has been retracted from the publication. We also tried to contact the authors, but did not receive a reply. The Editor apologizes to the readership for the inconvenience caused. [the original article was published in International Journal of Oncology 38: 973­983, 2011; DOI: 10.3892/ijo.2011.934]

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[Retracted] α3ß1 integrin promotes radiation-induced migration of meningioma cells.

Following the publication of the above paper, we were contacted by the University of Illinois at Chicago, to request the retraction of the above article. Following a formal institutional investigation, the investigation panel concluded that the images in question had falsifying elements. Regarding the above study, the specific allegations that were investigated were that of falsifying elements of Fig. 2A, right panel, row 3, columns 2, 3 and 4 and Fig. 4D, left panel, row 5, columns 1, 2 and 3; Fig. 4A, row 1, columns 2, 3 and 4, and Fig. 4C, row 1, columns 5, 6 and 7; and Fig. 6C, row 1, column 3, and row 2, column 1.
Following a review of this paper conducted independently by the Editor of International Journal of Oncology, the Editor concurred with the conclusions of the investigation panel, and therefore the above paper has been retracted from the publication. We also tried to contact the authors, but did not receive a reply. The Editor apologizes to the readership for the inconvenience caused. [the original article was published in International Journal of Oncology 40: 1615-1624, 2012; DOI: 10.3892/ijo.2011.987].