Chandramu Chetty

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.

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 3020: The role of NF-κB (p65) and Stat3 interplay in the regulation of radiation-induced, ICAM-1-mediated migration and invasion in glioma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Aberrant activation of both NF-κB and Stat3 is associated with enhanced tumor metastasis in various cancers. In human glioma xenograft cells 4910 and 5310, irradiation (IR; 8 Gy) elevated Stat3 and p65 phosphorylation and persistent nuclear translocation in correlation with massive increases in cellular and soluble ICAM-1 levels. Co-immunoprecipitation experiments confirmed enhanced nuclear phospho-p65/phospho-Stat3 and acetyl-p65/phospho-Stat3 interactions along with enhanced invasiveness and migration in IR-treated cells. Promoter analysis indicated presence of adjacent NF-κB- and Stat3-consensus binding elements in the proximal promoter and the proximal intron-1 of the ICAM-1 promoter, whereas overlapping NF-κB/Stat3 binding motifs were identified in the distal intron-1 region. ChIP binding and promoter/luciferase reporter assays showed that IR induced prominent binding of both Stat3 and NF-κB to the proximal intron-1 region followed by the distal intron-1 and the proximal promoter regions on the ICAM-1 gene. Individual treatments with Stat3.si or JSH-23 blocked the occupancy of both Stat3 and p65 onto the ICAM-1 promoter and prevented subsequent ICAM-1-mediated migration and invasion. In addition, constitutive activation of Stat3 and NF-κB by Stat3C or TNF-α significantly elevated IR-induced migration and invasion. In vivo orthotropic experiments in nude mice showed that the Stat3.si treatment reduced tumor growth and IR-induced phopsho-Stat3/phospho-p65 and ICAM-1 expression when compared to pSV-treated controls. These results suggest that both p65 and Stat3 bind to the intronic region of ICAM-1 promoter to induce its optimal transcription in IR-treated cells and abrogation of IR-induced p65/Stat3 binding inhibits ICAM-1-mediated enhancement of malignancy in post-irradiated tumor cells. As such, the results of this study clearly have potential future therapeutic significance in glioma treatment. 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 3020. doi:1538-7445.AM2012-3020

Abstract 2085: MMP-2 knockdown leads to anoikis and inhibits migration and invasion by attenuating αvβ3/EGFR/Pak4 survival signaling in human glioma xenograft cells in vitro and in vivo

MMP-2 plays a key role in the degradation of the extracellular matrix and thereby enhances the proliferative, invasive and metastatic potential of cancer cells. MMP-2 knockdown using siRNA (pM) in human glioma xenograft cell lines, 4910 and 5310, sensitized cells to anoikis associated with cleavage of PARP, and caspases 8 and 3. pM-inhibited cell adhesion, invasion and migration correlated with attenuation of EGFR, c-Src and Pak4 phosphorylation. High expression of Pak4 in human glioma cell lines and tumor tissues implicated its essential role in increasing glioma malignancy. Co-immunoprecipitation studies implied that MMP-2 directly interacts by complex formation with EGFR, p130CAS and Pak4 along with αvβ3 integrin and pM treatment disrupted this direct binding. Vitronectin-mediated αvβ3 activation and EGF and rhMMP-2 supplementation counteracted pM-inhibited, Pak4-mediated migration and invasion. Conversely, αvβ3 blocking antibody and ErbB-2/EGFR inhibitor (GW2974) further reduced pM-inhibited, Pak4-mediated invasiveness and migration, thereby suggesting a role of αvβ3 and EGFR in the regulation of Pak4 survival signaling. Individual treatments of pM and Pak4.si led to considerable anoikis in both 4910 and 5310 cells and simultaneous co-depletion MMP-2 and Pak4 resulted in robust increase in anoikis levels. In vivo orthotopic experiments in nude mice revealed decreased tumor size with pM treatment. Immunofluorescence analysis of pSV-tumor sections showed high expression and co-localization of MMP-2/αvβ3, which decreased upon treatment with pM along with significant decrease in phospho-EGFR, phospho-Pak4, Bcl2 and Bcl-xL levels. Our results imply a possible crosstalk between MMP-2 and Pak4 and suggest the potential of targeting both MMP-2 and Pak4 as a future therapeutic approach in glioma therapy. 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 2085. doi:1538-7445.AM2012-2085

Abstract 1391: Simultaneous knockdown of MMP-9 and uPAR/cathepsin B retards α9β1 integrin-mediated cell migration via SSAT and potassium channel pathway in glioma xenografts

The α9β1 integrin accelerates cell migration through the binding of α9 cytoplasmic domain to spermidine/spermine-N 1 -acetyltransferase (SSAT), which catalyzes the catabolism of higher order polyamines, spermidine and spermine, to the lower order polyamine, putrescine. Our recent studies have clearly demonstrated the role of α9β1 integrin in MMP-9, uPAR and cathepsin B-mediated cell migration in glioma xenograft cells (4910 and 5310). In the present study, we aimed to investigate the underlying mechanisms through which MMP-9 and uPAR/cathepsin B knockdown inhibits α9β1 integrin-mediated migration in glioma xenograft cells. Western blot, RT-PCR and immunofluorescence analyses revealed downregulation of SSAT levels both at the mRNA and protein levels by siRNA-mediated simultaneous knockdown of MMP-9 and uPAR/cathepsin B. In addition, we noted a prominent reduction in the expression of SSAT with MMP-9 and uPAR/cathepsin B knockdown in the tumor regions of 5310 injected nude mice brains. Co-localization of α9 integrin with kir4.2, an inward rectifier potassium channel, was observed in both 4910 and 5310 xenograft cells. However, MMP-9 and uPAR/cathepsin B knockdown in these cells prominently reduced the co-localization of α9 with kir4.2. Interestingly, MMP-9, uPAR and cathepsin B overexpression in these xenograft cells significantly elevated SSAT mRNA and protein levels. Further, the migratory potential of MMP-9/uPAR/cathepsin B overexpressed 4910 and 5310 cells was not affected by either glybenclamide or tertiapin-Q but instead was significantly inhibited by either barium or kir4.2 siRNA treatments. Taken together, our results from the present and earlier studies clearly demonstrate that α9β1 integrin-mediated cell migration utilizes SSAT and kir4.2 potassium channel pathway, and inhibition of the migratory potential of these glioma xenograft cells by simultaneous knockdown of MMP-9 and uPAR/cathepsin B could be attributed to the reduced SSAT levels and co-localization of α9 integrin with kir4.2 potassium channels. Based on these findings, we conclude that the simultaneous knockdown of MMP-9 and uPAR/cathepsin B retards SSAT and potassium channel pathway-mediated cell migration through the α9β1 integrin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1391. doi:10.1158/1538-7445.AM2011-1391

Abstract 4787: MMP-2 RNA interference leads to decreased NF-κB activation and induced JNK-mediated apoptosis by modulating TRADD-TRAF2-FADD interactions in glioma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL MMP-2 belongs to a family of extracellular matrix metalloproteinases (MMPs) that play a pivotal role in the degradation of the ECM, thereby enhancing the invasive and metastatic potential of cancer cells. Our study unveiled a new mechanism of how MMP-2 RNA interference inhibits NF-κB activation. Essentially, siRNA against MMP-2 (pM) modulated the interactions between the adaptor protein TRADD-TRAF2 and TRADD-FADD, which consequently hindered the nuclear localization and DNA binding activity of NF-κB. This, in turn, led to apoptosis. Furthermore, pM treatment led to RIP cleavage and remarkable increases in the expression levels of FADD and activated MAP kinases, p38 MAPK and JNK. The prolonged activation of JNK resulted in phosphorylation of c-Jun. Treatments with specific JNK1 inhibitor (SP600125) reduced and significantly reversed the pro-apoptotic effect of pM, thereby suggesting a role of JNK in pM-induced cell death. Supplementation of recombinant human MMP-2 to pM-transfected cells restored the inhibited DNA binding activity of NF-κB and rescued cells from the pro-apoptotic effect of pM. Here, we demonstrate that the simultaneous inhibition of NF-κB activation and sustained activation of JNK play significant roles in pM-mediated apoptosis in human xenograft glioma cells. In addition, our results show that the specific downregulation of MMP-2 diminished intracranial tumor growth in vivo. The inhibition of tumor growth after pM treatment was associated with decreased MMP-2 expression, inhibited NF-κB activation, and enhanced phospho-JNK levels. In conclusion, the present study elucidates an underlying mechanism of pM-induced cell death in vitro as well as decreased tumor development in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4787. doi:10.1158/1538-7445.AM2011-4787

Abstract 5503: Anti-proliferative role of MMP-9 and uPAR/cathepsin B knockdown in negative regulation of DNA repair mechanisms in human glioma xenografts

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Glioblastoma multiforme (GBM) is one of the most lethal forms of brain tumors. Efficient repair of DNA damage, especially double strand breaks (DSBs) and anti-apoptotic mechanisms associated with GBM, makes treating GBM challenging. In the present study, siRNA-mediated transcriptional silencing of MMP-9 and uPAR (MU) and MMP-9 and cathepsin B (MC) in 4910 and 5310 glioma xenograft cells had significant anti-proliferative effect as assessed by BrdU, MTT and clonogenic assays. FACS analysis revealed cells were in sub-G0-G1 phase after xenograft cells were treated with MU or MC. We also investigated the role of MU or MC knockdown in reducing DNA damage response (DDR), which eventually leads to apoptosis in glioma xenograft cells. RT-PCR and Western blot analyses revealed downregulation of DNA PKcs and Ku70/80 levels, which are responsible for non-homologous end joining (NHEJ) DNA repair. Elevated γH2AX protein expression and increased comet tail length indicated that the reduced NHEJ DNA repair led to accumulation of DSBs in MU and MC knocked down cells. The binding efficiency of Ku70 and Ku80 to DSB was significantly reduced in glioma cells treated with MU or MC. In addition, we noticed a reduced γH2AX and Ku70/Ku80 colocalization at DSBs in MU- and MC-treated xenograft cells. Moreover, MU and MC knockdown in these xenograft cells resulted in the downregulation of several other proteins involved in DNA damage response and the survival pathway (e.g., ATM, Rad 51, CHK2, EGFR, Akt, and ERK). Immunohistochemistry of the brain sections of xenografts injected into nude mice showed a reduced expression of DNA PKcs in animals treated with MU and MC siRNA. Taken together, our results clearly demonstrate that silencing via MU and MC not only negatively regulates DNA damage response, but also downregulates the proteins associated with the survival pathway. Based on these findings, it appears that silencing MU or MC in addition to radiation could be an effective strategy to treat radioresistant high-grade gliomas, where treatment usually fails due to the enhanced repair of DSBs induced by radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5503. doi:10.1158/1538-7445.AM2011-5503

Abstract 2286: SPARC stimulates neuronal differentiation of medulloblastoma cells via the IL-6/STAT3 pathway

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Secreted Protein Acidic and Rich in Cysteine (SPARC), a 37-kDa glycoprotein, participates in the regulation of morphogenesis and cellular differentiation through its modulation of cell-matrix interactions. We have reported previously that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we show that the ectopic expression of SPARC induces neuronal differentiation in medulloblastoma cells. Adenovirus-mediated expression of SPARC cDNA (Ad-DsRed-SP) elevated the expression of neuronal markers NeuN, Nestin and MAP-2 in medulloblastoma cells. SPARC expression decreased STAT3 phosphorylation and constitutive expression of STAT3 reversed SPARC-mediated neuronal differentiation. Moreover, our results show that SPARC expression not only inhibited interleukin (IL)-6 but also attenuated IL-6-mediated STAT3 phosphorylation in medulloblastoma cells. Taken together, our results suggest that SPARC induced neuronal differentiation of medulloblastoma tumor cells through its inhibitory effect on IL-6-mediated STAT3 signaling. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed increased staining for the neuronal markers NeuN, Nestin and MAP-2. Overall, our results demonstrate that SPARC promotes neuronal differentiation and inhibits tumor growth, thereby further supporting the potential use of SPARC as a therapeutic target for medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2286.

Abstract 2291: Downregulation of uPA/uPAR inhibits epithelial to mesenchymal transition (EMT) in intermittent hypoxic DAOY and D283 medulloblastoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Medulloblastoma is the most common, rapidly growing tumor of the cerebellum, which arises in childhood and has a tendency to metastasize via cerebrospinal fluid (CSF) pathways. In addition to surgical resection, post-operative radiotherapy and chemotherapy are the mainstays in treating patients with medulloblastoma. However, these tumors remain among the most challenging pediatric brain tumors. Factors related to the tumor microenvironment are being recognized as fundamental to tumor progression, increased resistance and metastasis. Hypoxia, one of these factors, leads to epigenetic and genetic adaptation of clones and increased EMT, invasiveness and metastasis. Thus, hypoxia is closely associated with tumorigenesis and provides tumors the ability to outgrow the existing vasculature. In this study, we show that intermittent hypoxia has a more pronounced effect than chronic hypoxia and is responsible for EMT, invasion, migration, and survival of hypoxic-conditioned medulloblastoma cells. We also show that Akt and SNAIL are activated under hypoxia, and this response is blocked by plasmid vector-mediated delivery of uPA/uPAR siRNA. The linkage of uPAR to Akt activation under hypoxia provides one mechanism by which uPAR may regulate SNAIL, and thus, promote EMT. The results presented here suggest that, in cells that express high levels of uPAR, induction and maintenance of EMT depends on simultaneous activation of numerous cell-signaling factors downstream of uPAR. This study provides valuable evidence of the anti-tumor efficacy of plasmid vector-mediated delivery of uPA/uPAR siRNA in hypoxic-conditioned medulloblastoma cells to target intermittent hypoxia-induced cell invasion, migration, angiogenesis and EMT and to achieve better therapeutic outcomes in the treatment of malignant medulloblastomas Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2291.