MCPIP1 expression positively correlates with melanoma-specific survival of patients, and its overexpression affects vital intracellular pathways of human melanoma cells.

The suppressive activity of monocyte chemoattractant protein 1-induced protein 1 (MCPIP1) an inflammation-related ribonuclease, has been described in a few cancer types but has yet to be assessed in the most common subtype of skin cancer: melanoma. Here, we have evaluated the MCPIP1 expression in melanoma tissues by reanalysis of publicly available transcriptome data from 89 melanoma samples, and immunohistochemical staining of 21 primary and 81 metastatic melanomas. Our data implicated decreased MCPIP1 expression in melanoma tumors compared to normal tissues, and positive correlation between high ribonuclease expression and melanoma-specific survival of patients. To investigate the ribonuclease activity in melanoma cells, MCPIP1 was ectopically expressed in the MV3 human melanoma cell line. Following the transcriptome, proteome, and intracellular signaling of MCPIP1-overexpressing MV3 cells was assessed via real-time quantitative polymerase chain reaction, Western blot analysis, and RNAseq. MV3 cells overexpressing MCPIP1 exhibited a broad range of alterations in the transcriptome and proteome, as well as in the phosphorylation status of a number of proteins, strongly indicating MCPIP1-dependent cell cycle arrest and inhibition of Akt/mTOR signaling in these cells. Moreover, we have shown, that MCPIP1 overexpression downregulates miRNA-193a-3p expression in MV3 cells. Furthermore, the majority of the described effects were dependent on the ribonucleolytic activity of the protein. The presented body of data strongly suggests a potential tumor suppressor role and possible future application as a positive prognostic marker of MCPIP1 protein in melanoma.

MCPIP1 ribonuclease can bind and cleave AURKA mRNA in MYCN-amplified neuroblastoma cells.

The role of the inflammation-silencing ribonuclease, MCPIP1 (monocyte chemoattractant protein-induced protein 1), in neoplasia continuous to emerge. The ribonuclease can cleave not only inflammation-related transcripts but also some microRNAs (miRNAs) and viral RNAs. The suppressive effect of the protein has been hitherto suggested in breast cancer, clear cell renal cell carcinoma, osteosarcoma, and neuroblastoma. Our previous results have demonstrated a reduced levels of several oncogenes, as well as inhibited growth of neuroblastoma cells upon MCPIP1 overexpression. Here, we investigate the mechanisms underlying the suppression of MYCN proto-oncogene, bHLH transcription factor (MYCN)-amplified neuroblastoma cells overexpressing the MCPIP1 protein. We showed that the levels of several transcripts involved in cell cycle progression decreased in BE(2)-C and KELLY cells overexpressing MCPIP1 in a ribonucleolytic activity-dependent manner. However, RNA immunoprecipitation indicated that only AURKA mRNA (encoding for Aurora A kinase) interacts with the ribonuclease. Furthermore, the application of a luciferase assay suggested MCPIP1-dependent destabilization of the transcript. Further analyses demonstrated that the entire conserved region of AURKA seems to be indispensable for the interaction with the MCPIP1 protein. Additionally, we examined the effect of the ribonuclease overexpression on the miRNA expression profile in MYCN-amplified neuroblastoma cells. However, no significant alterations were observed. Our data indicate a key role of the binding and cleavage of the AURKA transcript in an MCPIP1-dependent suppressive effect on neuroblastoma cells.

MCPIP1 overexpression in human neuroblastoma cell lines causes cell-cycle arrest by G1/S checkpoint block.

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) has a multidomain structure, which assures its pleiotropic activity. The physiological functions of this protein include repression of inflammatory processes and the prevention of immune disorders. The influence of MCPIP1 on the cell cycle of cancer cells has not been sufficiently elucidated. A previous study by our group reported that overexpression of MCPIP1 affects the cell viability, inhibits the activation of the phosphoinositide-3 kinase/mammalian target of rapamycin signalling pathway, and reduces the stability of the MYCN oncogene in neuroblastoma (NB) cells. Furthermore, a decrease in expression and phosphorylation levels of cyclin-dependent kinase (CDK) 1, which has a key role in the M phase of the cell cycle, was observed. On the basis of these previous results, the purpose of our present study was to elucidate the influence of MCPIP1 on the cell cycle of NB cells. It was confirmed that ectopic overexpression of MCPIP1 in two human NB cell lines, KELLY and BE(2)-C, inhibited cell proliferation. Furthermore, flow cytometric analyses and imaging of the cell cycle with a fluorescence ubiquitination cell-cycle indicator test, demonstrated that overexpression of MCPIP1 causes an accumulation of NB cells in the G1 phase of the cell cycle, while the possibility of an increase in G0 phase due to induction of quiescence or senescence was excluded. Additional assessment of the molecular machinery responsible for the transition between the cell-cycle phases confirmed that MCPIP1 overexpression reduced the expression of cyclins A2, B1, D1, D3, E1, and E2 and decreased the phosphorylation of CDK2 and CDK4, as well as retinoblastoma protein. In conclusion, the present results indicated a relevant impact of overexpression of MCPIP1 on the cell cycle, namely a block of the G1/S cell-cycle checkpoint, resulting in arrest of NB cells in the G1 phase.

GD2 ganglioside-binding antibody 14G2a and specific aurora A kinase inhibitor MK-5108 induce autophagy in IMR-32 neuroblastoma cells.

The process of autophagy and its role in survival of human neuroblastoma cell cultures was studied upon addition of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (14G2a mAb) and an aurora A kinase specific inhibitor, MK-5108. It was recently shown that combination of these agents significantly potentiates cytotoxicity against IMR-32 and CHP-134 neuroblastoma cells in vitro, as compared to the inhibitor used alone. In this study we gained mechanistic insights on autophagy in the observed cytotoxic effects exerted by both agents using cytotoxicity assays, RT-qPCR, immunoblotting, and autophagy detection methods. Enhancement of the autophagy process in the 14G2a mAb- and MK-5108-treated IMR-32 cells was documented by assessing autophagic flux. Application of a lysosomotropic agent-chloroquine (CQ) affected the 14G2a mAb- and MK-5108-stimulated autophagic flux. It is our conclusion that the 14G2a mAb (40 µg/ml) and MK-5108 inhibitor (0.1 µM) induce autophagy in IMR-32 cells. Moreover, the combinatorial treatment of IMR-32 cells with the 14G2a mAb and CQ significantly potentiates cytotoxic effect, as compared to CQ used alone. Most importantly, we showed that interfering with autophagy at its early and late step augments the 14G2a mAb-induced apoptosis, therefore we can conclude that inhibition of autophagy is the primary mechanism of the CQ-mediated sensitization to the 14G2a mAb-induced apoptosis. Although, there was no virtual stimulation of autophagy in the 14G2a mAb-treated CHP-134 neuroblastoma cells, we were able to show that PHLDA1 protein positively regulates autophagy and this process exists in a mutually exclusive manner with apoptosis in PHLDA1-silenced CHP-134 cells.

MCPIP1 Exogenous Overexpression Inhibits Pathways Regulating MYCN Oncoprotein Stability in Neuroblastoma.

The main physiological function of MCPIP1 (regnase-1) is negative regulation of inflammation. Moreover, roles of regnase-1 in apoptosis and differentiation have also been described, but its involvement in cancer is yet to be fully recognized. Earlier, we showed a lack of expression of MCPIP1 in both primary tumors and several neuroblastoma cell lines. Additionally, we reported that levels of MCPIP1 and the key neuroblastoma oncoprotein-MYCN were inversely correlated in BE(2)-C clones overexpressing the MCPIP1 gene. Here, we show that exogenous expression of the MCPIP1 protein decreases MYCN mRNA and protein levels without changing the MYCN mRNA half-life. Furthermore, it was shown that MCPIP1-wt exogenous expression affects levels and phosphorylation of MYCN partners such as Aurora A (Thr288), CDC2 (Tyr15 and Thr161), GSK3ß (Ser9), and key cellular components of Akt/mTOR signaling, which regulate MYCN stability and activation. In accordance with the obtained results, we found increased phosphorylation of MYCN protein at Thr58 that causes destabilization of the oncoprotein. Moreover, it is shown that exogenous expression of MCPIP1 does not cause apoptosis. Our data extend knowledge on roles of MCPIP1 in our model and link the protein to regulation of expression and stability of MYCN through decrease of signaling via Akt/mTOR pathway. J. Cell. Biochem. 118: 1741-1755, 2017. © 2016 Wiley Periodicals, Inc.

Structural Basis of GD2 Ganglioside and Mimetic Peptide Recognition by 14G2a Antibody.

Monoclonal antibodies targeting GD2 ganglioside (GD2) have recently been approved for the treatment of high risk neuroblastoma and are extensively evaluated in clinics in other indications. This study illustrates how a therapeutic antibody distinguishes between different types of gangliosides present on normal and cancer cells and informs how synthetic peptides can imitate ganglioside in its binding to the antibody. Using high resolution crystal structures we demonstrate that the ganglioside recognition by a model antibody (14G2a) is based primarily on an extended network of direct and water molecule mediated hydrogen bonds. Comparison of the GD2-Fab structure with that of a ligand free antibody reveals an induced fit mechanism of ligand binding. These conclusions are validated by directed mutagenesis and allowed structure guided generation of antibody variant with improved affinity toward GD2. Contrary to the carbohydrate, both evaluated mimetic peptides utilize a "key and lock" interaction mechanism complementing the surface of the antibody binding groove exactly as found in the empty structure. The interaction of both peptides with the Fab relies considerably on hydrophobic contacts however, the detailed connections differ significantly between the peptides. As such, the evaluated peptide carbohydrate mimicry is defined primarily in a functional and not in structural manner.

Monocyte Chemoattractant Protein-Induced Protein 1 Overexpression Modulates Transcriptome, Including MicroRNA, in Human Neuroblastoma Cells.

The recently discovered MCPIP1 (monocyte chemoattractant protein-induced protein 1), a multidomain protein encoded by the MCPIP1 (ZC3H12A) gene, has been described as a new differentiation factor, a ribonuclease, and a deubiquitination-supporting factor. However, its role in cancer is poorly recognized. Our recent analysis of microarrays data showed a lack of expression of the MCPIP1 transcript in primary neuroblastoma, the most common extracranial solid tumor in children. Additionally, enforced expression of the MCPIP1 gene in BE(2)-C cells caused a significant decrease in neuroblastoma proliferation and viability. Aim of the present study was to further investigate the role of MCPIP1 in neuroblastoma, using expression DNA microarrays and microRNA microarrays. Transient transfections of BE(2)-C cells were used for overexpression of either wild type of MCPIP1 (MCPIP1-wt) or its RN-ase defective mutant (MCPIP1-ΔPIN). We have analyzed changes of transcriptome and next, we have used qRT-PCR to verify mRNA levels of selected genes responding to MCPIP1 overexpression. Additionally, protein levels were determined for some of the selected genes. The choline transporter, CTL1, encoded by the SLC44A1 gene, was significantly repressed at the specific mRNA and protein levels and most importantly this translated into a decreased choline transport in MCPIP1-overexpressing cells. Then, we have found microRNA-3613-3p as the mostly altered in the pools of cells overexpressing the wild type MCPIP1. Next, we analyzed the predicted targets of the miR-3613-3p and validated them using qRT-PCR and western blot. These results indicate that the expression of miR-3613-3p might be regulated by MCPIP1 by cleavage of its precursor form.

CXCL12/CXCR4 blockade by oncolytic virotherapy inhibits ovarian cancer growth by decreasing immunosuppression and targeting cancer-initiating cells.

Signals mediated by the chemokine CXCL12 and its receptor CXCR4 are involved in the progression of ovarian cancer through enhancement of tumor angiogenesis and immunosuppressive networks that regulate dissemination of peritoneal metastasis and development of cancer-initiating cells (CICs). In this study, we investigated the antitumor efficacy of a CXCR4 antagonist expressed by oncolytic vaccinia virus (OVV) against an invasive variant of the murine epithelial ovarian cancer cell line ID8-T. This variant harbors a high frequency of CICs that form multilayered spheroid cells and express the hyaluronan receptor CD44, as well as stem cell factor receptor CD117 (c-kit). Using an orthotopic ID8-T tumor model, we observed that i.p. delivery of a CXCR4 antagonist-expressing OVV led to reduced metastatic spread of tumors and improved overall survival compared with oncolysis alone. Inhibition of tumor growth with the armed virus was associated with efficient killing of CICs, reduced expression of ascitic CXCL12 and vascular endothelial growth factor, and decreases in i.p. numbers of endothelial and myeloid cells, as well as plasmacytoid dendritic cells. These changes, together with reduced recruitment of T regulatory cells, were associated with higher ratios of IFN-γ(+)/IL-10(+) tumor-infiltrating T lymphocytes, as well as induction of spontaneous humoral and cellular antitumor responses. Similarly, the CXCR4 antagonist released from virally infected human CAOV2 ovarian carcinoma cells inhibited peritoneal dissemination of tumors in SCID mice, leading to improved tumor-free survival in a xenograft model. Our findings demonstrate that OVV armed with a CXCR4 antagonist represents a potent therapy for ovarian CICs with a broad antitumor repertoire.

Targeting of tumor-associated gangliosides with antibodies affects signaling pathways and leads to cell death including apoptosis.

Gangliosides are a diverse group of sialic acid containing glycosphigolipids that are abundantly present in an outer plasma membrane of some cells. Biological roles of gangliosides and other lipids in cell fate regulation are being extensively studied. Gangliosides are well known to be involved in interactions between cells and in signal transduction to regulate growth, adhesion and motility. Moreover, many gangliosides are tumor-associated antigens over-expressed on several tumor types. As a result, monoclonal antibodies binding gangliosides can be used to diagnose, monitor and to treat cancer patients. In the review, we gather and discuss data of various research groups on direct cytotoxic effects elicited by several ganglioside-specific antibodies, which bind to GM2, N-acetyl-GM2, N-glycolyl-GM2, GM3, GD3, GD2, O-acetyl-GD2, without involvement of immunological mechanisms. Thus, in cultures of numerous human and mouse cancer cell lines, the antibodies were reported to cause morphological changes, aggregation and detachment of cells, inhibition of proliferation and cell death involving necrosis, apoptosis and oncosis-like mechanisms. Additionally, data on proteome alterations were reviewed that encompass, among others, changes in kinome (P38, JNK, c-MET, ERK1/2, PI3K, AKT, FAK, aurora A, B, C), protein levels of transcription factors (P53, MYCN, HSF1) and pro-apoptotic proteins (caspase 3, BAX). Next, we collected data on application of the antibodies to enhance cytotoxicity of chemotherapeutic drugs and small molecule inhibitors. Finally, further research perspectives on the topic are discussed.

Silencing of the PHLDA1 leads to global proteome changes and differentiation pathways of human neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor originating from the abnormal development of cells of the sympathoadrenal lineage of the neural crest. Targeting GD2 ganglioside (GD2), a glycolipid expressed on neuroblastoma cells, with GD2 ganglioside-recognizing antibodies affects several pivotal signaling routes that drive or influence the malignant phenotype of the cells. Previously performed gene expression profiling helped us to identify the PHLDA1 (pleckstrin homology-like domain family A member 1) gene as the most upregulated gene in the IMR-32 human neuroblastoma cells treated with the mouse 14G2a monoclonal antibody. Mass spectrometry-based proteomic analyses were applied to better characterize a role of PHLDA1 protein in the response of neuroblastoma cells to chimeric ch14.18/CHO antibody. Additionally, global protein expression profile analysis in the IMR-32 cell line with PHLDA1 silencing revealed the increase in biological functions of mitochondria, accompanied by differentiation-like phenotype of the cells. Moreover, mass spectrometry analysis of the proteins co-immunoprecipitated using anti-PHLDA1-specific antibody, selected a group of possible PHLDA1 binding partners. Also, a more detailed analysis suggested that PHLDA1 interacts with the DCAF7/AUTS2 complex, a key component of neuronal differentiation in vitro. Importantly, our results indicate that PHLDA1 silencing enhances the EGF receptor signaling pathway and combinatory treatment of gefitinib and ch14.18/CHO antibodies might be beneficial for neuroblastoma patients. Data are available via ProteomeXchange with the identifier PXD044319.

Reprogramming the tumor microenvironment leverages CD8+ T cell responses to a shared tumor/self antigen in ovarian cancer.

Tumor antigen-driven responses to weakly immunogenic self-antigens and neoantigens directly affect treatment efficacy following immunotherapy. Using orthotopically grown SV40 T antigen+ ovarian carcinoma in antigen-naive wild-type or TgMISIIR-TAg-Low transgenic mice expressing SV40 T antigen as a self-antigen, we investigated the impact of CXCR4-antagonist-armed oncolytic virotherapy on tumor progression and antitumor immunity. Immunostaining and single-cell RNA sequencing analyses of the peritoneal tumor microenvironment of untreated tumors in syngeneic wild-type mice revealed the presence of SV40 T antigen-specific CD8+ T cells, a balanced M1/M2 transcriptomic signature of tumor-associated macrophages, and immunostimulatory cancer-associated fibroblasts. This contrasted with polarized M2 tumor-associated macrophages, immunosuppressive cancer-associated fibroblasts, and poor immune activation in TgMISIIR-TAg-Low mice. Intraperitoneal delivery of CXCR4-antagonist-armed oncolytic vaccinia virus led to nearly complete depletion of cancer-associated fibroblasts, M1 polarization of macrophages, and generation of SV40 T antigen-specific CD8+ T cells in transgenic mice. Cell depletion studies revealed that the therapeutic effect of armed oncolytic virotherapy was dependent primarily on CD8+ cells. These results demonstrate that targeting the interaction between immunosuppressive cancer-associated fibroblasts and macrophages in the tolerogenic tumor microenvironment by CXCR4-A-armed oncolytic virotherapy induces tumor/self-specific CD8+ T cell responses and consequently increases therapeutic efficacy in an immunocompetent ovarian cancer model.

The effects of garlic-derived sulfur compounds on cell proliferation, caspase 3 activity, thiol levels and anaerobic sulfur metabolism in human hepatoblastoma HepG2 cells.

The aim of the present studies was to determine whether the mechanism of biological action of garlic-derived sulfur compounds in human hepatoma (HepG2) cells can be dependent on the presence of labile sulfane sulfur in their molecules. We investigated the effect of allyl sulfides from garlic: monosulfide, disulfide and trisulfide on cell proliferation and viability, caspase 3 activity and hydrogen peroxide (H(2)O(2)) production in HepG2 cells. In parallel, we also examined the influence of the previously mentioned compounds on the levels of thiols, glutathione, cysteine and cysteinyl-glycine, and on the level of sulfane sulfur and the activity of its metabolic enzymes: rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionase. Among the compounds under study, diallyl trisulfide (DATS), a sulfane sulfur-containing compound, showed the highest biological activity in HepG2 cells. This compound increased the H(2)O(2) formation, lowered the thiol level and produced the strongest inhibition of cell proliferation and the greatest induction of caspase 3 activity in HepG2 cells. DATS did not affect the activity of sulfurtransferases and lowered sulfane sulfur level in HepG2 cells. It appears that sulfane sulfur containing DATS can be bioreduced in cancer cells to hydroperthiol that leads to H(2)O(2) generation, thereby influencing transmission of signals regulating cell proliferation and apoptosis.

Apoptosis is responsible for the cytotoxic effects of anti-GD2 ganglioside antibodies and aurora A kinase inhibitors on human neuroblastoma cells.

In recent years, immunotherapy has been identified as an effective treatment method for high-risk neuroblastoma. A previous study demonstrated that an anti-GD2 ganglioside (GD2) mouse 14G2a monoclonal antibody (mAb) combined with a small molecule, i.e., an aurora A kinase inhibitor (MK-5108), significantly increased cytotoxicity against human neuroblastoma cells, as compared to monotherapy. This study aimed to demonstrate the mechanism of neuroblastoma cell death in vitro following the addition of an anti-GD2 human-mouse chimeric ch14.18/CHO mAb (presently used in clinics) and two aurora A inhibitors (MK-5108 and MK-8745). The effects of the aforementioned agents on neuroblastoma cells were determined by measuring the level of ATP, the level of apoptotic and necroptotic markers, and the activity of caspase 3/7. The results revealed that the ch14.18/CHO mAb decreased cellular ATP levels in the IMR-32 and CHP-134 neuroblastoma cell lines, similarly to the 14G2a mAb. Regarding ch14.18/CHO mAb treated IMR-32 cells, the observed cytotoxic effect was concomitant with induced caspase 3 cleavage, which indicated the induction of apoptosis in IMR-32 cells, but not in CHP-134 cells. Furthermore, the MK-5108 inhibitor induced apoptosis, as indicated by the increased cleavage of caspase 3 and increased activity of caspase 3/7. However, the presence of necroptosis was ruled out in MK-5108-treated IMR-32 and CHP-134 cells. In summary, the effects of the combination of ch14.18/CHO mAb and aurora A kinase inhibitors (MK-5108 and MK-8745) were shown to enhance apoptosis in IMR-32 cells compared to when used individually.

Targeting a mimotope vaccine to activating Fcgamma receptors empowers dendritic cells to prime specific CD8+ T cell responses in tumor-bearing mice.

A major challenge for inducing antitumor immune responses with native or modified tumor/self-Ags in tumor-bearing hosts relates to achieving efficient uptake and processing by dendritic cells (DCs) to activate immune effector cells and limit the generation of regulatory T cell activity. We analyzed the ability of therapeutic DC vaccines expressing a CD166 cross-reactive mimotope of the GD2 ganglioside, 47-LDA, to selectively expand adoptively transferred, tumor-specific T cells in NXS2 neuroblastoma tumor-bearing syngeneic mice. Before the adoptive cell transfer and DC vaccination, the tumor-bearing mice were lymphodepleted by nonmyeloablative total body irradiation or a myeloablative regimen that required bone marrow transplantation. The 47-LDA mimotope was presented to DCs either as a linear polypeptide in conjunction with universal Th epitopes or as a fusion protein with the murine IgG2a Fc fragment (47-LDA-Fcgamma2a) to deliver the antigenic cassette to the activating Fcgamma receptors. We demonstrate that immunization of adoptively transferred T cells in tumor-bearing mice with the 47-LDA mimotope expressed in the context of the activating Fc fusion protein induced higher levels of antitumor immune responses and protection than the 47-LDA polypeptide-DC vaccine. The antitumor efficacy of the therapeutic 47-LDA-Fcgamma2a-DC vaccine was comparable to that achieved by a virotherapy-associated cancer vaccine using a recombinant oncolytic vaccinia virus expressing the 47-LDA-Fcgamma2a fusion protein. The latter treatment, however, did not require total body irradiation or adoptive cell transfer and resulted in induction of antitumor immune responses in the setting of established tolerance, paving the way for testing novel anticancer treatment strategies.

Induction of cell death in ovarian cancer cells by doxorubicin and oncolytic vaccinia virus is associated with CREB3L1 activation.

We have demonstrated that oncolytic vaccinia virus synergizes with doxorubicin (DOX) in inducing immunogenic cell death in platinum-resistant ovarian cancer cells and increases survival in syngeneic and xenograft tumor models. However, the mechanisms underlying the virus- and doxorubicin-mediated cancer cell death remain unknown. In this study, we investigated the effect of the oncolytic virus and doxorubicin used alone or in combination on activation of the cytoplasmic transcription factor CREB3L1 (cyclic AMP [cAMP] response element-binding protein 3-like 1) in ovarian cancer cell lines and clinical specimens. We demonstrated that doxorubicin-mediated cell death in ovarian cancer cell lines was associated with nuclear translocation of CREB3L1 and that the effect was augmented by infection with oncolytic vaccinia virus or treatment with recombinant interferon (IFN)-ß used as a viral surrogate. This combination treatment was also effective in mediating nuclear translocation of CREB3L1 in cancer cells isolated from ovarian tumor biopsies at different stages of disease progression. The measurement of CREB3L1 expression in clinical specimens of ovarian cancer revealed lack of correlation with the stage of disease progression, suggesting that understanding the mechanisms of nuclear accumulation of CREB3L1 after doxorubicin treatment alone or in combination with oncolytic virotherapy may lead to the development of more effective treatment strategies against ovarian cancer.

Transcription factors Elk-1 and SRF are engaged in IL1-dependent regulation of ZC3H12A expression.

BACKGROUND: MCPIP is a novel CCCH zinc finger protein described as an RNase engaged in the regulation of immune responses. The regulation of expression of the gene coding for MCPIP - ZC3H12A is poorly explored. RESULTS: Here we report that the proinflammatory cytokine IL-1beta rapidly induces the synthesis of MCPIP in primary monocyte-derived macrophages and HepG2 cells. This up-regulation takes place through the MAP kinase pathway and following activation of the transcription factor Elk-1. Using a ZC3H12A reporter construct we have shown that a ZC3H12A promoter region, stretching from -76 to +60, mediates activation by IL-1beta. This region contains binding sites for Elk-1 and its partner SRF. Chromatin immunoprecipitation analysis confirms in vivo binding of both transcription factors to this region of the ZC3H12A promoter. CONCLUSIONS: We conclude that the transcription factor Elk-1 plays an important role in the activation of ZC3H12A expression in response to IL-1beta stimulation.

Immunization with a mimotope of GD2 ganglioside induces CD8+ T cells that recognize cell adhesion molecules on tumor cells.

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.

EGF regulates plasminogen activator inhibitor-1 (PAI-1) by a pathway involving c-Src, PKCdelta, and sphingosine kinase 1 in glioblastoma cells.

Patients with gliomas expressing high levels of epidermal growth factor receptor (EGFR) and plasminogen activator inhibitor-1 (PAI-1) have a shorter overall survival prognosis. Moreover, EGF enhances PAI-1 expression in glioma cells. Although multiple known signaling cascades are activated by EGF in glioma cells, we show for the first time that EGF enhances expression of PAI-1 via sequential activation of c-Src, protein kinase C delta (PKCdelta), and sphingosine kinase 1 (SphK1), the enzyme that produces sphingosine-1-phosphate. EGF induced rapid phosphorylation of c-Src and PKCdelta and concomitant translocation of PKCdelta as well as SphK1 to the plasma membrane. Down-regulation of PKCdelta abolished EGF-induced SphK1 translocation and up-regulation of PAI-1 by EGF; whereas, down-regulation of PKCalpha had no effect on the EGF-induced PAI-1 activation but enhanced its basal expression. Similarly, inhibition of c-Src activity by PP2 blocked both EGF-induced translocation of SphK1 and PKCdelta to the plasma membrane and up-regulation of PAI-1 expression. Furthermore, SphK1 was indispensable for both EGF-induced c-Jun phosphorylation and PAI-1 expression. Collectively, our results provide a functional link between three critical downstream targets of EGF, c-Src, PKCdelta, and SphK1 that have all been implicated in regulating motility and invasion of glioma cells.

Recruitment of Intratumoral CD103+ Dendritic Cells by a CXCR4 Antagonist-Armed Virotherapy Enhances Antitumor Immunity.

Intratumoral dendritic cells play an important role in stimulating cytotoxic T cells and driving antitumor immunity. Using a metastatic ovarian tumor model in syngeneic mice, we explored whether therapy with a CXCR4 antagonist-armed oncolytic vaccinia virus activates endogenous CD103+ dendritic cell responses associated with the induction of adaptive immunity against viral and tumor antigens. The overall goal of this study was to determine whether expansion of CD103+ dendritic cells by the virally delivered CXCR4 antagonist augments overall survival and in situ boosting with a tumor antigen peptide-based vaccine. We found that locoregional delivery of the CXCR4-A-armed virus reduced the tumor load and the immunosuppressive network in the tumor microenvironment, leading to infiltration of CD103+ dendritic cells that were capable of phagocytic clearance of cellular material from virally infected cancer cells. Further expansion of tumor-resident CD103+ DCs by injecting the FMS-related tyrosine kinase 3 ligand, the formative cytokine for CD103+ DCs, provided a platform for a booster immunization with the Wilms tumor antigen 1 peptide-based vaccine delivered intraperitoneally with polyriboinosinic:polyribocytidylic acid as an adjuvant. The vaccine-induced antitumor responses inhibited tumor growth and increased overall survival, indicating that expansion of intratumoral CD103+ dendritic cells by CXCR4-A-armed oncovirotherapy treatment can potentiate in situ cancer vaccine boosting.

The effects of modulation of gamma-glutamyl transpeptidase activity in HepG2 cells on thiol homeostasis and caspase-3-activity.

The present studies aimed to elucidate how the modulation of gamma-glutamyl transpeptidase (gammaGT) activity in human hepatoma (HepG2) cell line influences H(2)O(2) production, caspase 3 activity, protein S-thiolation by glutathione (GSH), cysteinyl-glycine (Cys-Gly) and cysteine (Cys), and the level of other redox forms of these thiols. The experiments showed that 1-h stimulation of gammaGT elevated H(2)O(2) production, leading to prooxidant conditions. After 24-h stimulation, H(2)O(2) concentration was at the control level, while Cys-Gly-, Cys- and GSH-dependent S-thiolation was markedly increased, which was accompanied by a drop in caspase-3 activity. The inhibition of gammaGT activity by acivicin led to H(2)O(2) decrease after 1-h incubation which still persisted after 24 h. The inhibition of gammaGT activity in HepG2 cells was also connected with the lowering of S-thiolation with Cys and Cys-Gly and with increasing of caspase-3 activity. The results of our studies indicate that the modulation of gammaGT activity can be used to change cellular redox status, and can affect Cys- and Cys-Gly-dependent S-thiolation and caspase-3 activity. We suggest that the role of high gammaGT activity in HepG2 cells can be connected with production of reactive oxygen species and with S-thiolation with Cys and Cys-Gly that can influence activity of caspase 3.