Venkateswara Rao Gogineni

Irradiation-induced angiogenesis is associated with an MMP-9-miR-494-syndecan-1 regulatory loop in medulloblastoma cells

Matrix metalloproteinase-9 (MMP-9) represents one of the most prominent proteins associated with tumorigenesis and is a modulator of the tumor microenvironment during angiogenesis. Recently, syndecan-1 (SDC1), a transmembrane heparan sulfate-bearing proteoglycan, was also speculated to have a critical role in contributing to angiogenesis when associated with MMP-9. However, the mechanism behind their synergistic regulation is not fully understood. In the current study, we report for the first time that ionizing radiation (IR)-induced MMP-9 enhances SDC1 shedding, corroborating to tube-inducing ability of medulloblastoma (MB) cells. Furthermore, we observed that the tumor angiogenesis is associated with higher MMP-9–SDC1 interactions on both the cell surface and extracellular medium. Our results also revealed the existence of a novel regulatory mechanism where MMP-9 drives the suppression of miR-494, resulting in enhanced SDC1 shedding and angiogenesis. From the in situ hybridization analysis, we found that MMP-9-specific shRNA (shMMP-9) treatment of mouse intracranial tumors resulted in elevated expression of miR-494. This negative correlation between MMP-9 and miR-494 levels was observed to be dependent on the methylation status of a miR-494 promoter-associated CpG island region (−186 to −20), which was confirmed by bisulfite-sequencing and methylation-specific PCR (MSP) analysis. Further, validation of MMP-9 and SDC1 3′-untranslated region (3′-UTR) targets with luciferase reporter assay provided a more favorable result for miR-494-mediated regulation of SDC1 but not of MMP-9, suggesting that the 3′-UTR of SDC1 mRNA is a direct target of miR-494. Overall, our results indicate that angiogenesis induced by radiotherapy is associated with an MMP-9–miR-494–SDC1 regulatory loop and that MMP-9–SDC1 activity creates a negative feedback loop by regulating the expression of miR-494.

Chk2-mediated G2/M cell cycle arrest maintains radiation resistance in malignant meningioma cells

In continuation to our studies on radioresistance in meningioma, here we show that radiation treatment (7Gy) induces G2/M cell cycle arrest in meningioma cells. Phosphorylation of Chk2, Cdc25c and Cdc2 were found to be key events since interference with Chk2 activation and cyclin B1/Cdc2 interaction led to permanent arrest followed by apoptosis. Irradiated cells showed recovery and formed aggressive intracranial tumors with rapid spread and morbidity. Nevertheless, knock down of uPAR with or without radiation induced permanent arrest in G2/M phase and subsequent apoptosis in vitro and in vivo. In conclusion, our data suggest that combination treatment with radiation and uPAR knock down or other inhibitors resulting in non-reversible G2/M arrest may be beneficial in the management of meningiomas.

MMP-2 mediates mesenchymal stem cell tropism towards medulloblastoma tumors

Matrix metalloproteinases (MMPs) are a family of proteinases known to have a role in cell migration. In the present study, we evaluated the role of MMP-2 on tropism of human umbilical cord blood-derived stem cells (hUCBSCs) in a human medulloblastoma tumor model. Consequences of MMP-2 inhibition on stem cell tropism towards medulloblastoma were studied in terms of stem cell migration by using cell culture inserts, transwell chamber assay, western blotting for MMP-2 and migratory molecules, and immunohistochemistry. Conditioned medium from Daoy/D283 cells infected with adenoviral vector encoding MMP-2 small interfering RNA (siRNA) (Ad-MMP-2 si)-reduced stem cell migration as compared with conditioned medium from mock and scrambled vector (Ad-SV) infected cells. In addition, MMP-2 inhibition in the tumor cells decreased the expression of stromal cell-derived factor 1 (SDF1) in the tumor-conditioned medium, which results in impaired SDF1/CXCR4 signaling leading to decreased stem cell tropism towards the tumor cells. We further show that MMP-2 inhibition in the tumor cells repressed stem cell tropism towards medulloblastoma tumors in vivo. In summary, we conclude that hUCBSCs can integrate into human medulloblastoma after local delivery and that MMP-2 expression by the tumor cells mediates this response through the SDF1/CXCR4 axis.

uPAR and cathepsin B shRNA impedes TGF-β1-driven proliferation and invasion of meningioma cells in a XIAP-dependent pathway

Overexpression of transforming growth factor β1 (TGF-β1) has been linked to immune suppression, tumor angiogenesis, tumor cell migration, tumor cell survival, and tumor cell invasion in many cancers. In the present study, we found abundant expression of TGF-β1 in the microenvironment of four different pathological types of meningioma tumors. TGF-β1 induced invasion in malignant meningioma cells with an associated upregulation of urokinase-type plasminogen activator (uPA), uPAR, cathepsin B, and MMP-9, and this increase in proliferation was coupled with the expression of anti-apoptotic and pro-survival signaling molecules. In addition to the intense immunoreactivity of meningioma tumors to X-linked inhibitor to apoptosis (XIAP), its knockdown abolished the TGF-β1-induced proliferation of these cells. The stimulation of XIAP expression and the activation of pSMAD-2 is mediated by phosphatidylinositol 3-kinase (PI3K)- and MEK-dependent pathways, and the addition of anti-TGF-β1 antibodies prevented their expression with a consequent decrease in invasion. Bicistronic shRNA constructs targeting uPAR and cathepsin B (pUC) quenched TGF-β1-driven invasion and survival of meningioma cells by downregulation of XIAP and pSMAD-2 expression. Animal models with intracranial tumors showed elevated levels of TGF-β1, XIAP and pSMAD-2, and pUC treatment prevented this increased expression. Thus, targeted silencing of TGF-β1-induced signaling by pUC in meningioma would provide new treatment approaches for management of meningioma.

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.

The secreted protein acidic and rich in cysteine (SPARC) induces endoplasmic reticulum stress leading to autophagy-mediated apoptosis in neuroblastoma.

Our previous studies showed that overexpression of secreted protein acidic and rich in cysteine (SPARC) induced autophagy-mediated apoptosis in PNET cells. In the present study, we attempted to elucidate the molecular mechanisms and signaling cascades associated with SPARC overexpression in combination with radiation therapy that eventually leads to autophagy-mediated apoptosis in neuroblastoma. SPARC expression in SK-N-AS and NB-1691 cells demonstrated the activation of caspase 3, cleavage of PARP and induction of apoptosis. The experiments to unravel the mechanisms associated with autophagy-apoptosis illustrated that SPARC overexpression triggered endoplasmic reticulum (ER) stress and thereby unfolded protein response (UPR). This was apparent with the activation of stress receptors, inositol-requiring enzyme (IRE 1α), RNA-dependent protein kinase (PKR)-like ER kinase (PERK) and BiP. This study further demonstrated the induction of transcription factor CHOP as a result of IRE-JNK activation in response to increased SPARC levels. Inhibition of ER stress and JNK activation led to inhibition of autophagy-mediated apoptosis. Further, the apparent expression of ER stress molecules among the orthotopic tumors treated by SPARC overexpression plasmids substantiated our in vitro observations. Taken together, these results illustrate the critical role of ER stress in regulating autophagy-mediated apoptosis in SPARC-overexpressed neuroblastoma cells and radiation treatment.

Suppression of uPA and uPAR blocks radiation-induced MCP-1 mediated recruitment of endothelial cells in meningioma.

Chemokines play a vital role in recruiting various cell types in the process of tissue repair. Radiation, a major therapeutic modality in cancer treatment, has been described to induce inflammatory response that might lead to the expression of several chemokines. In the present study, we investigated the mechanism of monocyte chemoattractant protein-1 (MCP-1) induction by radiation in meningioma cell lines and the paracrine effect on human microvascular endothelial cells (HMEC). After radiation, meningioma cell lines (IOMM Lee and SF-3061) showed an increased expression of MCP-1. In addition, irradiated meningioma cancer cell conditioned medium (CM) showed an increased ability to attract HMEC and to stimulate MCP-1-induced protein (MCPIP), VEGF and angiogenin expression in HMEC. This chemotactic activity and angiogenic stimulator effect on HMEC were almost abrogated by depleting MCP-1 from the irradiated cancer cell CM. Further, inhibition of either ERK activation/expression or NF-κB nuclear translocation hindered radiation-induced MCP-1 expression in both meningioma cell lines. Further, supplementing cancer cells with exogenous ATF-uPA (with and without radiation) activated ERK phosphorylation, nuclear translocation of the NF-κB p65 sub-unit (Rel-A), and MCP-1 expression. Downregulation of uPA and uPAR, simultaneously by transfecting the cancer cells with bi-cistronic siRNA-expressing plasmid (pU) inhibited radiation-induced ERK activation, nuclear translocation of Rel-A, NF-κB DNA binding activity, and MCP-1 expression. In addition, pU-transfected cancer cells (with or without radiation) reduced radiation-induced MCP-1 and blocked the recruitment of other cell types during the inflammatory process induced by radiation both in in vitro and in vivo conditions.

uPAR/cathepsin B overexpression reverse angiogenesis by rescuing FAK phosphorylation in uPAR/cathepsin B down regulated meningioma.

BACKGROUND Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Surgery and radiation therapy is a common treatment for brain tumors, however, patients whose tumors recur after such treatments have limited therapeutic options. Earlier studies have reported important roles of uPA, uPAR and cathepsin B in tumor progression. METHODOLOGY/PRINCIPAL FINDINGS In the present study, we examined the therapeutic significance of RNAi-mediated simultaneous down regulation of these proteolytic networks using two bicistronic siRNA constructs, pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation in two different meningioma cell lines. Transfection of meningioma cells with pUC and pU2 significantly reduced angiogenesis as compared to control treatment both in vitro and in vivo nude mice model. This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF). Expression of focal adhesion kinase (FAK) is elevated in malignant meningioma, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment. In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation. Transient over-expression of uPAR and cathepsin B by full length uPAR/cathepsin B (FLpU/C) in pUC transfected meningioma cells promoted vascular phenotype, rescued expression of Ang-1, Ang-2, VEGF, FAK (Y925) and Grb2 both in vitro and in vivo mice model. CONCLUSION/SIGNIFICANCE These studies provide the first direct proof that bicistronic siRNA construct for uPAR and cathepsin B (pUC) reduces Y925-FAK activity and this inhibition is rescued by overexpression of both uPAR and cathepsin B which clearly demonstrates that pUC could thus be a potential therapeutic approach as an anti-angiogenic agent in meningioma.

α3β1 Integrin Promotes Radiation-induced Migration of Meningioma Cells

Cell motility is influenced by the microenvironment, signal transduction and cytoskeleton rearrangement. Cancer cells become resistant to these control mechanisms and gain the ability to move throughout the body and invade healthy tissues, which leads to metastatic disease. Integrins respond to context-dependent cues and promote cell migration and survival in cancer cells. In the present study, we analyzed the role of integrins in radiation-induced migration of meningioma cells. Migration and cell proliferation assays revealed that radiation treatment (7 Gy) significantly increased migration and decreased proliferation in two cell lines, IOMM-Lee and CH-157-MN. α3 and β1 integrins were overexpressed at both the protein and transcript levels after radiation treatment and a function-blocking α3β1 antibody inhibited the radiation-induced migration. Immunofluorescence studies illustrated the localization of α3 integrin and F-actin at the migration front of irradiated cells. Further, an increase in phosphorylation of FAK and ERK was observed, while both FAK phosphorylation inhibitor and FAK shRNA inhibited ERK phosphorylation and downregulated uPA and vinculin. In addition to the co-localization of FAK and ERK at the migration front, these FAK-inhibition results link the downstream effects of ERK to FAK. Correspondingly, U0126 quenched ERK phosphorylation and reduced the expression of molecules involved in migration. Furthermore, brain sections of the animals implanted with tumors followed by radiation treatment showed elevated levels of α3 integrin and active ERK. Taken together, our results show that radiation treatment enhances the migration of meningioma cells with the involvement of α3β1 integrin-mediated signaling via FAK and ERK.

Fed‐batch cultivation of Escherichia coli expressed designer hepatitis C virus diagnostic intermediate and its evaluation

The present study aimed for an enhanced induction strategy combined with high‐level production of a capture antigen of hepatitis C virus (HCV) for use in diagnosis of HCV infection. We have expressed the synthetic gene encoding for HCV multiepitope protein in pET‐28a(+) vector and investigated its production in Escherichia coli BL21(DE3) cells using high‐cell‐density fed‐batch cultivation. A maximum cell dry mass of 30 g/L was obtained, and the culture was induced with 1, 5, and 10 mM isopropyl β‐d‐1‐thiogalactopyranoside (IPTG) for ∼4 H at 30°C; a maximum protein production of 1.5 g/L was observed in the case of induction with 10 mM IPTG. The enhanced induction strategy resulted in a ∼15‐fold increase as compared to 1 mM IPTG. The protein was purified using a simple immobilized metal affinity chromatography procedure, yielding 16.6 mg/g dry cell weight of pure protein with more than 99% purity. Further, the protein was evaluated for its diagnostic potential by using the commercially available HCV Seroconversion Panel, Worldwide HCV Performance Panel, and Viral Coinfection Panel. The protein showed high sensitivity and specificity, which was comparable to the best performing commercially available enzyme immunoassay (EIA) kits.