Jasti S. Rao

Functional cooperativity by direct interaction between PAK4 and MMP-2 in the regulation of anoikis resistance, migration and invasion in glioma

Gliomas display anoikis resistance, enhanced invasion in to the adjacent brain parenchyma and eventually recur despite using the standard therapies. Our studies on increased anoikis sensitization in matrix metalloproteinase-2 (MMP-2)-knockdown 4910 and 5310 human glioma xenograft cells were interestingly correlated with p21-activated kinase 4 (PAK4) inhibition, prompting us to further investigate the role of PAK4 in glioma. Here, we report the PAK4 upregulation in positive correlation with increasing glioma pathological grades. The siRNA-mediated PAK4 knockdown elevated anoikis, and inhibited invasion and migration by downregulating MMP-2, αvβ3-integrin and phospho-epidermal growth factor receptor (phospho-EGFR). The cDNA-PCR arrays revealed a transcriptional suppression of essential proteins involved in cell proliferation and adhesion in PAK4-knockdown cells. Most importantly, glutathione S-transferase pull-down assays demonstrated the MMP-2 as a new PAK4-interacting protein which binds to PAK4 kinase domain. Individual EGFR/ErbB2 inhibitor and αvβ3 antibody treatments in PAK4si-treated cells indicated the regulation of αvβ3/EGFR survival signaling by PAK4. Overexpression of PAK4 significantly reversed the MMP2si-induced cell death in both cell lines. Codepletion of PAK4 and MMP-2 resulted in robust anoikis-mediated cell death, and severely inhibited invasive and migratory properties in these cells. PAK4si inhibited in vivo tumor growth in nude mice by inhibiting MMP-2, β3-integrin and phospho-EGFR levels in tumors. Our findings indicate a physical association between PAK4 and MMP-2, and suggest the future therapeutic potential of PAK4/MMP-2 dual targeting in glioma treatment.

Mesenchymal Stem Cell Therapy for Apoptosis After Spinal Cord Injury

Spinal cord injury (SCI) is a devastating clinical problem that has irreversible consequences, results in permanent functional loss, and life time disability (Sekhon and Fehlings, 2001). This debilitating condition often affects young and healthy individuals at the prime of their life, creates enormous physical and emotional cost, and places a significant financial burden to society at large (Ackery et al., 2004). Even though years of research have led to a better understanding in the pathophysiology of permanent neural injuries at the cellular level, much of the mechanism and processes of secondary injury at the molecular level remain to be elucidated. With modern molecular strategies and techniques, breakthroughs in the understanding of neuronal injury and neural regeneration provide new promises for reversal of spinal cord injury that once was thought to be permanent and irreversible (Carlson and Gorden, 2002). Spinal cord injury involves an initial mechanical or primary injury followed by a series of cellular and molecular secondary events that amplify the extent of the initial damage and results in the progressive destruction of spinal cord tissue. After acute contusion, the spinal cord undergoes a sequential progression of pathologic changes, including micro hemorrhage, cytotoxic edema, neuronal necrosis, axonal fragmentation, demyelination, further secondary cellular destruction and eventually cyst formation (Balentine, 1978; Balentine and Greene, 1984; Coutts and Keirstead, 2008). Damage to the spinal cord results in extensive proliferation of microglia and macrophages in and around the injury epicenter. This acute inflammatory response at the injury site is at least partly responsible for secondary spinal cord injury ( Popovich et al., 1997; Carlson et al., 1998; Taoka et al., 1998). The inflammatory cells (particularly macrophages/microglia) mediate tissue damage by producing a variety of cytotoxic factors including interleukins (Rice et al., 2007) and tumor necrosis factor-alpha (TNF-α) (Beattie et al., 2002). White matter breakdown begins at the grey-white matter junction with progressive edema (Dohrmann et al., 1972). Axoplasmic stasis and axonal swelling that contains multiple organelles, mitochondria, neurofilament, and smooth endoplasmic reticulum eventually undergo glandular dissolution and myelin

Abstract 2635: Galectin-1 modulates glioblastoma cell migration via interaction with FAK.

Malignant glioblastomas (GBM) are the most common brain tumors and exhibit excessive growth, cell cycle regulation, neovascularization, angiogenesis, migration, immune escape and resistance to apoptosis. Galectin-1 (Gal-1) is a 14.5-kDa β-galactoside-binding protein, which displays intracellular (i.e., protein-protein interactions) and extracellular (i.e., protein-oligosaccharide interactions) functions with marked pro-angiogenic and pro-migratory effects in gliomas. Although glioma is considered one of the deadliest cancers with median survival ranging from nine to twelve months, therapeutic treatments that target Gal-1 may prove effective since Gal-1 is one of the genes that cause aggressive behaviors of human GBM such as migration and invasion. Gal-1 controls glioma cell migration and invasion by modifying the actin cytoskeleton and expression of small GTPases. In the present study, immunohistochemical, Real Time PCR and Western blot experiments performed with clinical samples demonstrated that Gal-1 is highly expressed in human GBM tissue as compared to healthy brain tissue. Matrigel invasion assays confirmed that treatment of glioma cell lines U251 and 5310 with human umbilical cord blood stem cells (hUCBSC) almost completely blocked invasion of GBM cells. We also observed that treatment of GBM cells with hUCBSC decreased the expression of both Gal-1 and FAK. Additionally, silencing either the Gal-1 or FAK gene using shRNA decreased migration and invasion of glioblastoma cells. Furthermore, using immunocytochemistry and Western blot analyses, we observed that Gal-1 and FAK interact with each other in glioblastoma. Taken together, our results suggest that Gal-1 and FAK might have a direct protein-protein interaction for their function in the tumorigenesis of glioblastoma. Citation Format: Kiran Kumar Velpula, Arnima Bhasin, Jane S. Zhang, Andrew J. Tsung, Krishna Kumar Veeravalli, Jasti S. Rao, Venkata Ramesh Dasari. Galectin-1 modulates glioblastoma cell migration via interaction with FAK. [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 2635. doi:10.1158/1538-7445.AM2013-2635

Abstract 5187: Urokinase plasminogen activator receptor (uPAR) and integrin α5β1 regulate plasminogen activator inhibitor-1 (PAI-1) activity in medulloblastoma cells

The urokinase plasminogen activator (uPA) system is a dynamic extracellular protease system that regulates both proteolytic and non-proteolytic events associated with cancer progression. In earlier studies, we have demonstrated that radiation-induced cell adhesion was associated with uPAR expression and downregulation of uPAR effectively inhibited radiation-induced cell adhesion of medulloblastoma cells. Herein, we further investigated the impact of uPAR downregulation on plasminogen activator inhibitor (PAI-1), a key regulator component of the uPA system, and its role in cell adhesion. With radiation treatment, the levels of uPAR and PAI-1 increased in D283 and UW228 cells. Surprisingly, knockdown of uPAR elevated the levels of PAI-1. The increase in PAI-1 levels in uPAR-knocked down cells was regulated by activation of cJUN/CREB signaling molecules. Moreover, studies show that the reduced interaction between uPAR and integrin α5β1 plays a critical role in PAI-1 release into the extracellular matrix, thereby enhancing the cell detachment process. Incubation of medulloblastoma cells with Tiplaxtinin-PAI-039, a potent PAI-1 activity inhibitor, increased the association of uPAR with integrin α5β1 and cell adhesion. Interestingly, prolonged inhibition of PAI-1 activity adversely affected cell viability; we were able to revert this effect by exogenously supplementing cells with recombinant PAI-1. Moreover r-PAI-1 showed no significant effect on cell adhesion whereas it increased the association of uPAR with integrin α5β1. Expression of full-length (FL) uPAR elevated uPA and PAI-1 levels both at the transcriptional and the translational levels. These increased PAI-1 levels play a pivotal role in regulating uPA activity by binding and translocating integrin α5β1 towards the uPA/uPAR complex. Taken together, the results of the present study lead us to conclude that levels of uPAR and integrin α5β1, and their association on the cell surface dictate the function of PAI-1 to maintain extracellular homeostasis. 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 5187. doi:1538-7445.AM2012-5187

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 3376: Cord blood stem cells revert EMT to MET in glioma stem cells by downregulating synergistic transcriptional activation of Sox2 and Twist1

The dynamic nature of cancer stem cells that underlie metastasis or their ability to switch between different cellular identities, as in epithelial to mesenchymal transition and mesenchymal to epithelial transition has profound implications for cancer therapy. The functional relationship between molecules involved in cancer cell stemness and metastasis is not clear. In this regard, our studies on human glioblastoma tissue grade IV specimens showed significant expression of Twist1 and Sox2, known mesenchymal and stemness related markers, respectively, indicating their association with glial tumorigenesis and metastasis. The glioma stem cells obtained from CD133 + cells demonstrated increased expression of Twist1 and Sox2 accompanied by significant increase in the mesenchymal markers such as N-cadherin, vimentin and β-catenin. Our studies on glioma stem cells treatment with human umbilical cord blood derived mesenchymal stem cells, showed down regulation of Twist1 and Sox2 proteins, apart from other mesenchymal stem cell markers. Based on the in vitro experiments and in vivo intracranial xenograft mouse model studies we elucidated the potential therapeutic role of hUCBSC in suppressing glioma cancer stemness by the induction of mesenchymal to epithelial transition. 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 3376. doi:1538-7445.AM2012-3376

Abstract 461: Involvement of α9β1 integrin-iNOS pathway in matrix metalloproteinase-9 and/or urokinase plasminogen activator receptor-mediated glioma cell migration

Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide (NO) production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Our recent studies have clearly demonstrated the role of α9β1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we aimed to investigate the involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. Immunohistochemical analysis of the glioblastoma multiforme (GBM) tissue array revealed the prominent presence of iNOS in several clinical GBM samples. In addition, immunofluorescence analysis of U251 glioma cell lines and 5310 glioma xenograft cells showed a prominent expression of iNOS. Transcriptional inactivation of MMP-9 and/or uPAR by respective shRNA reduced iNOS expression in both U521 and 5310 glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in these glioma cells after transfection with MMP-9- or uPAR-overexpressing plasmids. Wound healing and spheroid migration assays showed a significant inhibition of the migration potential of MMP-9- or uPAR-overexpressed U251 glioma cells after treatment with L-NAME, an inhibitor of iNOS. Similarly, Matrigel invasion assay revealed a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed U251 and 5310 glioma cells after L-NAME treatment. Further, we noticed a prominent reduction of iNOS expression in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Integrin α9β1 regulates iNOS activity via Src tyrosine kinase; Src coordinates subsequent signaling pathways through activation of FAK and tyrosine phosphorylation of the adaptor protein p130Cas. Western blot analysis of MMP-9 and/or uPAR shRNA treated U251 and 5310 glioma cells revealed significant reductions in the protein expression of cSrc, phosphoSrc, FAK and p130Cas after simultaneous downregulation of both MMP-9 and uPAR. Taken together, our results from the present and earlier studies clearly demonstrate that α9β1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of these glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9β1 integrin and iNOS levels. Therefore, our results highlight the possible involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. 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 461. doi:1538-7445.AM2012-461

Abstract 1118: Protease-integrin regulates mitochondrial biogenesis in glioma xenograft cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Various studies have described mitochondrial dysfunction and its association with several diseases, including cancer. Regulation of mitochondrial biogenesis via mitochondrial-nuclear genome encoded protein signaling has been identified in recent literature. In the present study, we explored whether matrix metalloproteinase-9 (MMP-9) shRNA mediates mitochondrial biogenesis and alters glioma proliferation. We found that MMP-9 shRNA initiated mitochondrial biogenesis in glioma cells by increasing the expression of mitochondrial transcription factor (mtTFA). MMP-9 silencing elevated nuclear respiratory factor-1 (NRF1) and -2 (NRF2), which are involved in transcription regulation of mtTFA. Mitochondrial localization of mtTFA was more evident in MMP-9-treated glioma cells as compared to untreated cells. In addition, MMP-9 suppression significantly increased the binding of mtTFA to light strand and heavy strand promoter of mitochondrial genome as determined by mitochondrial chromatin immunoprecipitation assay. MMP-9 transcriptional suppression decreased the expression of phospho Akt and PI3K while increasing PTEN expression in glioma xenograft cells. We also show that dominant negative Akt increased mtTFA, NRF1 and NRF2, and decreased PI3K, integrin αv and integrin β3. Further, MMP-9 and Akt suppression increased the nuclear and mitochondrial expression of mtTFA. As indicated by increased copy number of displacement (D) loop, mitochondrial replication was observed in both MMP-9- and Akt-downregulated glioma cells. As confirmed by immunoprecipitation analysis, interaction of αvβ3 integrin with MMP-9 decreased. Studies with functional blocking of αvβ3 integrin indicated elevation of mtTFA, a decrease in phospho Akt, and overexpression of D-loop; these data indicate MMP-9 mediates mitochondrial biogenesis via αvβ3 integrin-Akt/PTEN, which led to increased mtTFA expression. Earlier studies from our lab also showed apoptosis induction in these glioma cells by MMP-9 shRNA. Moreover, 4910 and 5310 glioma xenograft tissue sections from mice treated with MMP-9 shRNA showed reduced expression of phospho Akt, and elevated PTEN and mtTFA levels. Decreased co-localization of αvβ3 integrin and MMP-9 was associated with MMP-9-suppressed tumor sections. Further, RT-PCR and western blot analyses showed increased expression of proteins involved in mitochondrial biogenesis encoded both by mitochondrial and nuclear genome in MMP-9 shRNA-treated glioma cells. Elevated levels of reactive oxygen species and the results of the FACS analysis show increased mitochondrial organelle stain in MMP-9-silenced glioma cells. Taken together, these data provide new insight into MMP-9-mediated glioma proliferation inhibition by mitochondrial biogenesis induced cell death. 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 1118. doi:1538-7445.AM2012-1118

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 2218: Radiation-induced hypomethylation triggers urokinase plasminogen activator (uPA) transcription in meningioma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Our previous studies have shown the role of radiation-induced urokinase plasminogen activator (uPA) expression in meningioma tumor progression. In the present study, we investigated whether modulation of DNA methylation profiles could regulate uPA expression. Radiation treatment induced hypomethylation in meningioma cells with a decrease in DNMT1 and MBD expression. However, oxidative damage by H2O2 or pretreatment of irradiated cells with NAC did not show any influence on these proteins, thereby indicating a radiation-specific change in the methylation patterns among meningioma cells. Further, we identified that hypomethylation is coupled to an increase in uPA expression in these cells. Azacytidine treatment induced a dose-dependent surge of uPA expression while pretreatment with Sodium butyrate inhibited the radiation-induced uPA expression; these results complement our prior findings. Methylation-specific PCR on bisulfite treated genomic DNA revealed a diminished methylation of uPA promoter in irradiated cells, which supports the role of epigenetics in uPA gene regulation. Transfection with shRNA expressing plasmids targeting CpG islands of the uPA promoter showed a marked decline in uPA expression with subsequent decreases in invasion and proliferation of meningioma cells. Further, radiation treatment is coupled to the recruitment of SP1 transcription into the nuclear extracts, which was abrogated by shRNA treatment. Our experiments to study the signaling events demonstrated the activation of MEK-ERK in radiation treated cells while U0126 (MEK/ERK inhibitor) blocked hypomethylation, recruitment of SP1, and uPA expression. In agreement with our in vitro data, low DNMT1 levels and high uPA were found in intracranial tumors given radiation treatment as compared to untreated tumors. In conclusion, our data suggest radiation-mediated hypomethylation triggers uPA expression in meningioma 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 2218. doi:1538-7445.AM2012-2218