Divya Kesanakurti

MMP-2 siRNA Inhibits Radiation-Enhanced Invasiveness in Glioma Cells

Background Our previous work and that of others strongly suggests a relationship between the infiltrative phenotype of gliomas and the expression of MMP-2. Radiation therapy, which represents one of the mainstays of glioma treatment, is known to increase cell invasion by inducing MMP-2. Thus, inhibition of MMP-2 provides a potential means for improving the efficacy of radiotherapy for malignant glioma. Methodology/Principal Findings We have tested the ability of a plasmid vector-mediated MMP-2 siRNA (p-MMP-2) to modulate ionizing radiation-induced invasive phenotype in the human glioma cell lines U251 and U87. Cells that were transfected with p-MMP-2 with and without radiation showed a marked reduction of MMP-2 compared to controls and pSV-transfected cells. A significant reduction of proliferation, migration, invasion and angiogenesis of cells transfected with p-MMP-2 and in combination with radiation was observed compared to controls. Western blot analysis revealed that radiation-enhanced levels of VEGF, VEGFR-2, pVEGFR-2, p-FAK, and p-p38 were inhibited with p-MMP-2-transfected cells. TUNEL staining showed that radiation did not induce apoptosis in U87 and U251 cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to controls. Intracranial tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation compared to controls. Conclusion/Significance MMP-2 inhibition, mediated by p-MMP-2 and in combination with radiation, significantly reduced tumor cell migration, invasion, angiogenesis and tumor growth by modulating several important downstream signaling molecules and directing cells towards apoptosis. Taken together, our results demonstrate the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in glioma patients.

Role of MMP-2 in the regulation of IL-6/Stat3 survival signaling via interaction with α5β1 integrin in glioma.

Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2′-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-α, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment.

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.

Suppression of MMP-2 attenuates TNF-α induced NF-κB activation and leads to JNK mediated cell death in glioma.

Background Abrogation of apoptosis for prolonged cell survival is essential in cancer progression. In our previous studies, we showed the MMP-2 downregulation induced apoptosis in cancer cell lines. Here, we attempt to investigate the exact molecular mechanism of how MMP-2 depletion leads to apoptosis in glioma xenograft cell lines. Methodology/Principal Findings MMP-2 transcriptional suppression by MMP-2siRNA (pM) induces apoptosis associated with PARP, caspase-8 and -3 cleavage in human glioma xenograft cells 4910 and 5310. Western blotting and cytokine array showed significant decrease in the cellular and secreted levels of TNF-α with concomitant reduction in TNFR1, TRADD, TRAF2, RIP, IKKβ and pIκBα expression levels resulting in inhibition of p65 phosphorylation and nuclear translocation in pM-treated cells when compared to mock and pSV controls. In addition MMP-2 suppression led to elevated Fas-L, Fas and FADD expression levels along with increased p38 and JNK phosphorylation. The JNK-activity assay showed prolonged JNK activation in pM-transfected cells. Specific inhibition of p38 with SB203580 did not show any effect whereas inhibition of JNK phosphorylation with SP600125 notably reversed pM-induced cleavage of PARP, caspase-8 and -3, demonstrating a significant role of JNK in pM-induced cell death. Supplementation of rhMMP-2 counteracted the effect of pM by remarkably elevating TNF-α, TRADD, IKKβ and pIκBα expression and decreasing FADD, Fas-L, and phospho-JNK levels. The EMSA analysis indicated significant reversal of pM-inhibited NF-κB activity by rhMMP-2 treatment which rescued cells from pM-induced cell death. In vivo studies indicated that pM treatment diminished intracranial tumor growth and the immuno histochemical analysis showed decreased phospho-p65 and enhanced phospho-JNK levels that correlated with increased TUNEL-positive apoptotic cells in pM-treated tumor sections. Conclusion/Significance In summary, our study implies a role of MMP-2 in the regulation of TNF-α mediated constitutive NF-κB activation and Fas-mediated JNK mediated apoptosis in glioma xenograft cells in vitro and in vivo.

MMP-2 suppression abrogates irradiation-induced microtubule formation in endothelial cells by inhibiting αVβ3-mediated SDF-1/CXCR4 signaling

The majority of glioblastoma multiforme (GBM) tumors recur after radiation (IR) treatment due to increased angiogenesis and IR-induced signaling events in endothelial cells (ECs) that are involved in tumor neovascularization; however, these signaling events have yet to be well characterized. In the present study, we observed that IR (8 Gy) significantly elevated MMP-2 expression and gelatinolytic activity in 4910 and 5310 human GBM xenograft cells. In addition, ECs treated with tumor-conditioned media (CM) obtained from IR-treated 4910 and 5310 cells showed increased microtubule formation. In view of this finding, we investigated the possible anti-angiogenic effects of MMP-2 downregulation using siRNA (pM.si) in IR-treated cells. We also determined the effect of CM obtained from mock, pSV (scrambled vector) and pMMP-2.si on endothelial cell growth and vessel formation. pM.si-CM-treated ECs showed inhibited IR-CM-induced SDF-1, CXCR4, phospho-PI3K and phospho-AKT and αvβ3 expression levels. In vitro angiogenesis assays also showed that the pM.si+IR decreased IR-induced vessel formation in ECs. Immunofluorescence and immunoprecipitation experiments indicated the abrogation of αvβ3-SDF-1 interaction in pM.si-CM-treated ECs when compared to mock or pSV treatments. External supplementation of either rhMMP-2 or rhSDF-1 counteracted and noticeably reversed pM.si-inhibited SDF-1, CXCR4, phospho-PI3K and phospho-AKT expression levels and angiogenesis, thereby confirming the role of MMP-2 in the regulation of αvβ3-mediated SDF-1/CXCR4 signaling. In addition to the in vitro results, the in vivo mouse dorsal air sac model also showed reduced angiogenesis after injection of pM.si alone or in combination with IR-treated xenograft cells. In contrast, injection of mock or pSV-treated cells resulted in robust formation of characteristic neovascularization. Collectively, our data demonstrate the role of MMP-2 in the regulation of SDF-1/CXCR4 signaling-mediated angiogenesis in ECs and show the anti-angiogenic efficacy of combining MMP-2 downregulation and IR when treating patients with GBM in the future.

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.

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 1031: Blockade of radiation-induced neuropilin-1 in glioblastoma cells impairs migration of endothelial cells

Neuropilin-1 (NRP-1) is an isoform specific receptor for vascular endothelial growth factor (VEGF), that regulates the development of the nervous system and also promotes angiogenesis in endothelial cells. Glioblastoma multiforme (GBM) is a malignant tumor characterized by robust neoangiogenesis with an aberrant elevation of VEGF. Recent evidence suggests that the majority of GBMs after radiation therapy (IR) exhibit an angiogenic regeneration process, which contributes phenotypic plasticity and therapeutic resistance. A high expression of NRP-1 in GBM correlates with disease progression. In this study, we show that the IR-induced NRP-1 role in the VEGFR-2 mediated signaling cascade promotes migration of endothelial cells. We observed that IR (8Gy) significantly elevated levels of VEGF and NRP-1 expression in 4910 and 5310 human GBM xenograft cells. Endothelial cells cultured on tumor- conditioned media from IR induced xenograft cells showed a significant increase in migration of endothelial cells; whereas, conditioned medium (CM) from NRP-1 knockdown xenograft cells inhibited IR-induced migration effects in endothelial cells. Further, CM from NRP-1 inhibited cells downregulated IR-induced expression of VEGFR-2 and its downstream molecules Sema3A, Plexin-A1 and E-cadherin in endothelial cells. Immunoflourescence co-localization studies revealed that the interaction of NRP-1 and VEGFR-2 were significantly decreased in endothelial cells cultured on CM from NRP-1-siRNA-transfected xenograft cells compared to mock- and SV-transfected cells. Collectively, our data indicate that NRP-1 plays a significant role in the regulation of VEGF/VEGFR-2 signaling and migration in endothelial cells, and thereby suggests NRP-1 as an important therapeutic target for glioma patients receiving radiation therapy. Citation Format: Dilip Rajasekhar Maddirela, Divya Kesanakurti, Venkateswara R. Gogineni, Chandramu Chetty. Blockade of radiation-induced neuropilin-1 in glioblastoma cells impairs migration of endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1031. doi:10.1158/1538-7445.AM2014-1031

Abstract 3070: Suppression of tumor cell invasiveness and in vivo tumor growth by microRNA-874 in non-small cell lung cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC MicroRNAs are a family of small non-coding RNAs that regulate expression of many 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. Interestingly, in silico target prediction analysis revealed numerous pro-oncogenes associated with tumor initiation, angiogenesis, invasion and migration, including MMP-2, uPA, NFAT5, PAK3, PAK7, LIMD, PECAM1, VEGF-A, Stat3, CUX1 and FGF1 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 as compared to its normal counterparts. Overexpression of miR-874 in CD133-positive NSCLC cancer stem cell (CSC) population led to a significant loss in CSC phenotype and enhanced sphere de-differentiation into epithelial-like morphology. Restoration of miR-874 expression drastically reduced the invasive ability of cells in comparison to mock and control-miR treated cells by suppressing the protein levels of MMP-2 and uPA. Further, miR-874 treatment decreased orthotopic tumor growth in nude mice as compared to mock and control-miR treatments. Further, in vivo orthotopic tumor experiments in nude mice revealed the significant decrease in the immunoreactivity of human anti-MMP-2 and anti-uPA in miR-874-treated tumor sections. 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 future treatment of NSCLC. Citation Format: Divya Kesanakurti, Dilip Maddirela, Subramanyam Chittivelu, Jasti S. Rao, Chandramu Chetty. Suppression of tumor cell invasiveness and in vivo tumor growth by microRNA-874 in non-small cell lung cancer. [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 3070. doi:10.1158/1538-7445.AM2013-3070 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5100: MMP-2 suppression inhibits α5β1 integrin and IL-3-mediated Stat5 activation and proliferation of endothelial cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Endothelial cell (EC) proliferation is a major component of angiogenesis in glioblastoma multiforme (GBM) and is regulated by several growth factors including IL-3. In this study, we have evaluated the effect of conditioned media (CM) obtained from MMP-2 knockdown xenograft 4910 and 5310 cell lines by pMMP-2 siRNA (pM.si) on the growth of ECs. Western blot analysis of CM from pM.si-treated 4910 and 5310 cells showed a significant downregulation of MMP-2 and IL-3 expression compared to mock- and pSV (scrambled vector)-CM. Proliferation decreased in ECs cultured on pM.si-CM from the xenograft cells when compared to ECs cultured on mock- or pSV-CM as determined by Ki-67 staining and MTT assays. Since Stat5 is a known target of IL-3, induces EC proliferation, and promotes neo-angiogenesis, we examined the role of pM.si in regulating IL-3-mediated Jak2/Stat5 signaling in ECs. Jak2/Stat5 suppression in ECs when cultured on pM.si-CM resulted in the decreased expression of integrin β1, Stat5, Bcl2 and Cyclin D2 at the transcription level as compared with ECs cultured on mock- and pSV-CM, as confirmed by RT-PCR analysis. Supplementation of recombinant human IL-3 counteracted and noticeably reversed pM.si-inhibited IL-3Rα, Jak2, p-Stat5 expression and proliferation of ECs, thereby confirming the role of MMP-2 in the regulation of α5β1/IL-3 mediated Jak2/Stat5 signaling. Immunoprecipitation and immunofluorescence studies revealed MMP-2 knockdown resulted in the decreased interaction of MMP-2 and integrin α5β1 in ECs, thereby implicating the downregulation of Jak2/Stat5 expression. Immunohistochemical analysis in tumor sections corroborated our in vitro studies, suggesting that the expression of p-Stat5 and Bcl2 are significantly decreased upon pM.si treatment compared to mock or pSV treatments. Our results suggest that MMP-2 knockdown by pM.si can lead to a significant inhibition of IL-3-induced proliferation of ECs and can be an effective anti-angiogenic therapy when treating patients with GBM in the future. Citation Format: Dilip Rajasekhar Maddirela, Divya Kesanakurti, Chandramu Chetty, Jasti S. Rao. MMP-2 suppression inhibits α5β1 integrin and IL-3-mediated Stat5 activation and proliferation of endothelial 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 5100. doi:10.1158/1538-7445.AM2013-5100