Novel read through agent: ZKN-0013 demonstrates efficacy in APCmin model of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is a precancerous, colorectal disease characterized by hundreds to thousands of adenomatous polyps caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). Approximately 30% of these mutations are premature termination codons (PTC), resulting in the production of a truncated, dysfunctional APC protein. Consequently, the ß-catenin degradation complex fails to form in the cytoplasm, leading to elevated nuclear levels of ß-catenin and unregulated ß-catenin/wnt-pathway signaling. We present in vitro and in vivo data demonstrating that the novel macrolide, ZKN-0013, promotes read through of premature stop codons, leading to functional restoration of full-length APC protein. Human colorectal carcinoma SW403 and SW1417 cells harboring PTC mutations in the APC gene showed reduced levels of nuclear ß-catenin and c-myc upon treatment with ZKN-0013, indicating that the macrolide-mediated read through of premature stop codons produced bioactive APC protein and inhibited the ß-catenin/wnt-pathway. In a mouse model of adenomatous polyposis coli, treatment of APCmin mice with ZKN-0013 caused a significant decrease in intestinal polyps, adenomas, and associated anemia, resulting in increased survival. Immunohistochemistry revealed decreased nuclear ß-catenin staining in the epithelial cells of the polyps in ZKN-0013-treated APCmin mice, confirming the impact on the ß-catenin/wnt-pathway. These results indicate that ZKN-0013 may have therapeutic potential for the treatment of FAP caused by nonsense mutations in the APC gene. KEY MESSAGES: • ZKN-0013 inhibited the growth of human colon carcinoma cells with APC nonsense mutations. • ZKN-0013 promoted read through of premature stop codons in the APC gene. • In APCmin mice, ZKN-0013 treatment reduced intestinal polyps and their progression to adenomas. • ZKN-0013 treatment in APCmin mice resulted in reduced anemia and increased survival.

P2Y12 receptor expression is a critical determinant of functional responsiveness to ATX's MORFO domain.

In the central nervous system, the formation of the myelin sheath and the differentiation of the myelinating cells, namely oligodendrocytes, are regulated by complex signaling networks that involve purinergic receptors and the extracellular matrix. However, the exact nature of the molecular interactions underlying these networks still needs to be defined. In this respect, the data presented here reveal a signaling mechanism that is characterized by an interaction between the purinergic P2Y(12) receptor and the matricellular extracellular matrix protein autotaxin (ATX), also known as ENPP2, phosphodiesterase-Iα/ATX, or lysoPLD. ATX has been previously described by us to mediate intermediate states of oligodendrocyte adhesion and to enable changes in oligodendrocyte morphology that are thought to be crucial for the formation of a fully functional myelin sheath. This functional property of ATX is mediated by ATX's modulator of oligodendrocyte remodeling and focal adhesion organization (MORFO) domain. Here, we show that the expression of the P2Y(12) receptor is necessary for ATX's MORFO domain to exert its effects on differentiating oligodendrocytes. In addition, our data demonstrate that exogenous expression of the P2Y(12) receptor can render cells responsive to the known effects of ATX's MORFO domain, and they identify Rac1 as an intracellular factor mediating the effect of ATX-MORFO-P2Y(12) signaling on the assembly of focal adhesions. Our data further support the idea that a physical interaction between ATX and the P2Y(12) receptor provides the basis for an ATX-MORFO-P2Y(12) signaling axis that is crucial for mediating cellular states of intermediate adhesion and morphological/structural plasticity.

Contrasting effects of interleukin-2 secretion by rat glioma cells contingent upon anatomical location: accelerated tumorigenesis in the central nervous system and complete rejection in the periphery.

Rat T9.F glioma cells were transduced with the interleukin (IL)-2 gene. Clone T9.F/IL2/#12 secreted a high level of IL-2 (15 ng/10(6) cells/48 h). Enhanced tumor progression and reduced survival was observed when T9.F/IL2/#12 cells were implanted intracranially. Subcutaneous injection of T9.F/IL2/#12 cells induced a palpable nodule, which regressed in approximately 15 days, resulting in tumor-specific protection. Lymphocytes from T9.F/IL2/#12 primed rats specifically respond to T9.F antigens but lacked cytotoxicity towards T9.F cells. Intracranial T9.F/IL2/#12 tumors were markedly infiltrated by CD4(+) and CD8(+) T cells, natural killer (NK)-T cells and myeloid progenitor cells, whereas subcutaneous T9.F/IL2/#12 tumors contained an elevated level of NK cells.

17α-androstenediol-mediated oncophagy of tumor cells by different mechanisms is determined by the target tumor.

Δ5-androstene-3ß,17α-diol (17α-AED) mediates oncophagy of human myeloid, glioma, and breast tumor cells by apoptotic- and autophagic-programmed cell death pathways, whereas the 17ß-epimer does not. In hematologically derived myeloid tumor cells, 17α-AED induced apoptosis, as determined by TUNEL staining, caspase, PARP activation, and electron microscopy. In contrast, 17α-AED treatment of glioma cells of neuroectodermal lineaged induced autophagy, evident by the presence of acidic vesicular organelles, LC3 processing, and upregulation of beclin-1. Proliferation inhibition studies on primary and established glioma cells demonstrated that the IC-50 of the steroid is ∼15 µM. In the case of breast cancer cells, the bioactivity of 17α-AED is independent of the expression of estrogen or androgen receptors. Collectively, oncophagy is induced by 17α-AED treatment in human tumor cells and proceeds by the induction of either autophagy or apoptosis. The neoplastic cell determines which oncophagic pathway is utilized.

Tumor-infiltrating, myeloid-derived suppressor cells inhibit T cell activity by nitric oxide production in an intracranial rat glioma + vaccination model.

In rats bearing an intracranial T9 glioma, immunization with tumor antigens induces myeloid suppressor cells, which express neutrophil (His48) and monocyte (CD11bc) markers, to infiltrate the tumors. The His48(+)/CD11bc(+) cells were not derived from CNS microglia but were hematogenous; suppressed multiple T cell effector functions; and are myeloid-derived suppressor cells (MDSC). The glioma-infiltrating MDSC expressed arginase I, iNOS, indoleamine 2,3-dioxygenase and TGF-beta; however, inhibitor/blocking studies demonstrated that NO production was the primary mechanism of suppression which induced T cell apoptosis. These findings suggest that neuro-immunomodulation by MDSC in rat gliomas maybe mediated by a pathway requiring NO production.

The neuro-steroid, 5-androstene 3ß,17α diol; induces endoplasmic reticulum stress and autophagy through PERK/eIF2α signaling in malignant glioma cells and transformed fibroblasts.

In this study, we identified a mechanism by which the neuro-steroid, 5-androstene 3ß,17α diol (17α-AED) induces autophagy in human malignant glioma cells and transformed fibroblasts. 17α-AED treatment induced endoplasmic reticulum (ER) stress, identified by the partial activation of an unfolded protein response in T98G, U87MG, U251MG, LN-18, LN-229 and LN-Z308 glioma cell lines. In this regard, there were increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein of 78kDa transcripts but no splicing of X-box-binding protein 1 mRNA or processing of activating transcription factor-6 in glioma cells treated with the neuro-steroid. 17α-AED induced eukaryotic translational initiation factor 2α (eIF2α) phosphorylation in glioma cells which correlated with microtubule-associated protein-light chain 3 (LC3) conversion from LC3-I to -II. In transformed murine embryonic fibroblasts (MEFs) that are deficient of eIF2α function or T98G glioma cells transfected with a dominant-negative eIF2α construct, 17α-AED induced LC3 conversion was significantly reduced as compared to control cells. Neuro-steroid treatment caused the activation of the eIF2α kinase, protein kinase-like ER kinase (PERK) but not other eIF2α kinases in glioma cells. Moreover, eIF2α phosphorylation and LC3 conversion, in response to 17α-AED treatment, was blocked in MEFs that lacked PERK activity. T98G cells transfected with a dominant-negative PERK construct exhibited an attenuated response to neuro-steroid treatment in terms of decreases in: eIF2α activation; CHOP expression; the incidence of autophagy; and cytotoxicity. These results demonstrate that ER stress is linked to 17α-AED induced autophagy by PERK/eIF2α signaling in human malignant glioma cells and transformed fibroblasts.

Effect of WT1 gene silencing on the tumorigenicity of human glioblastoma multiforme cells.

OBJECT: Wilms tumor 1 (WT1) is overexpressed in many human cancers, including glioblastoma multiforme (GBM). In another study, the authors showed that transient WT1 silencing increases the radiosensitivity of glioma cells. Studies of nonglioma cell lines have demonstrated that WT1 promotes cell proliferation and survival; however, this ability has not been rigorously analyzed in human GBM. METHODS: The authors tested the efficacy of 2 sequences of short hairpin RNA (shRNA) directed against WT1 in U251MG human GBM cells and found that 1 sequence was capable of stably silencing WT1 expression. They then evaluated the effect of WT1 silencing on cellular proliferation, invasion, and in vivo tumor formation. RESULTS: Stable WT1-shRNA expression significantly decreased the proliferation of U251MG cells in vitro as demonstrated by both an adenosine 5'-triphosphate-based viability assay and tritiated thymidine uptake. Furthermore, stable WT1 silencing caused significantly slower growth after the subcutaneous inoculation of tumor cells in the flanks of athymic nude mice and was associated with an increased latency period. CONCLUSIONS: Data in this study provide proof of the principle that downregulation of WT1 causes decreased tumorigenicity of a GBM cell line in vitro and in vivo and suggest that WT1 is a promising target for novel molecular GBM therapies, perhaps in combination with standard treatment modalities.

The anti-tumor effects of androstene steroids exhibit a strict structure-activity relationship dependent upon the orientation of the hydroxyl group on carbon-17.

Androstene steroids are metabolites of dehydroepiandrosterone and exist as androstene-diols or -triols in alpha- and beta-epimeric forms based upon the placement of the hydroxyl groups relative to the plane of the Delta(5)cycloperhydrophenanthrene ring. 5-Androstene-3beta,17beta-diol (3beta,17beta-AED) functions to upregulate immunity and the addition of a third hydroxyl group at C-7 in the alpha- or beta-orientation (3beta,7alpha,17beta-AET and 3beta,7beta,17beta-AET, respectively) enhances the immunological activity of the molecule. In contrast, 5-androstene-3beta,17alpha-diol (3beta,17alpha-AED) possesses potent anti-tumor activity. We synthesized a new androstene by adding a third hydroxyl group at C-7 to make 5-androstene-3beta,7alpha,17alpha-triol (3beta,7alpha,17alpha-AET) and compared the anti-tumor activity of this steroid to the four existing androstenes. The results showed that this modification reduced the activity of 3beta,17alpha-AED. The ranking of the anti-tumor activities of these steroids and their IC50 on human glioblastoma and lymphoma cells was: 3beta,17alpha-AED ( approximately 10 microm) > 3beta,7alpha,17alpha-AET ( approximately 30 microm) " 3beta,7alpha,17beta-AET ( approximately 150 microm)> 3beta,7beta,17beta-AET (not achievable) >or= 3beta,17beta-AED (not achievable). 3beta,17alpha-AED and 3beta,7alpha,17alpha-AET induced autophagy in T98G glioblastoma cells and apoptosis in U937 lymphoma cells. These results indicate that the position of the hydroxyl group on C-17 dictates the anti-tumor activity of the androstenes and must be in the alpha-configuration, demonstrating a strict structure-activity relationship.

Autophagy and the functional roles of Atg5 and beclin-1 in the anti-tumor effects of 3beta androstene 17alpha diol neuro-steroid on malignant glioma cells.

In this study, we demonstrate that the anti-tumor activity of the neuro-steroid, 3beta androstene 17alpha diol (17alpha-AED) on malignant glioma cells is mediated by the induction of autophagy. 17alpha-AED can inhibit the proliferation an induce cell death of multiple, unrelated gliomas with an IC(50) between 8 and 25muM. 17alpha-AED treatment induced the formation of autophagosomes and acidic vesicular organelles in human malignant gliomas which was blocked by bafilomycin A1 or 3-methyladenine. Cleavage of microtubule-associated protein-light chain 3 (LC3), an essential step in autophagosome formation, was detected in human malignant glioma cells exposed to 17alpha-AED. In 17alpha-AED treated T98G glioma cells there was an increase in the autophagy related proteins Atg5 and beclin-1. Silencing of ATG5 or beclin-1 with small interfering RNA significantly reduced the incidence of autophagy in 17alpha-AED treated malignant gliomas and attenuated the cytotoxic effects of the neuro-steroid indicating that the induction of autophagy mediates the anti-glioma activity of 17alpha-AED rather than serving as a cyto-protective response. These results demonstrate that 17alpha-AED possesses significant anti-glioma activity when used at pharmacologically relevant concentrations in vitro and the cytotoxic effects are resultant from the induction of autophagy.

FTY720 reduces inflammation and promotes functional recovery after spinal cord injury.

A robust and complex inflammatory cascade is known to be a prominent component of secondary injury following spinal cord injury (SCI). Specifically, the concept of trauma-induced autoimmunity has linked the lymphocyte population with neural tissue injury and neurologic deficit. FTY720, a sphingosine receptor modulator that sequesters lymphocytes in secondary lymphoid organs, has been shown to be effective in the treatment of a variety of experimental autoimmune disorders. Accordingly, by reducing lymphocyte infiltration into the spinal cord following SCI, this novel immunomodulator may enhance tissue preservation and functional recovery. In the present study, a moderate to severe contusion SCI was simulated in adult Long-Evans hooded rats. Using flow cytometry we showed that daily FTY720 treatment dramatically reduced T-cell infiltration into the SCI lesion site at 4 and 7 days post-injury, while other inflammatory cell populations were relatively unaltered. To assess functional recovery, three groups of injured animals (treated, vehicle, and injury only) were evaluated weekly for hindlimb recovery. Animals in the treated group consistently exhibited higher functional scores than animals in the control groups after 2 weeks post-injury. This finding was associated with a greater degree of white matter sparing at the lesion epicenter when cords were later sectioned and stained. Furthermore, treated animals were found to exhibit improved bladder function and a reduced incidence of hemorrhagic cystitis compared to control counterparts. Collectively these results demonstrate the neuroprotective potential of FTY720 treatment after experimental SCI.

Down-regulation of Wilms' tumor 1 expression in glioblastoma cells increases radiosensitivity independently of p53.

The Wilms' tumor 1 (WT1) gene is overexpressed in human glioblastoma and correlates with wild-type p53 status. In other cell types, WT1 inhibits p53-mediated apoptosis in response to DNA damaging agents. However, neither this interaction nor the relationship between WT1 and radiosensitivity has been studied in glioblastoma. To study this interaction, we generated LN-229 glioma cell lines (p53 mutant) stably expressing WT1 isoforms and induced apoptosis by transfecting with different doses of wild-type p53 plasmid expression vector. Constitutive expression of WT1 did not protect against exogenous p53-mediated apoptosis. Likewise, WT1 expression did not protect against endogenous p53-mediated cell death induced by radiotherapy in U87MG cells, which contain functional wild-type p53. We then tested the efficacy of WT1 siRNA in inhibiting WT1 expression and its effect on radiosensitivity. In T98G and LN-18 glioma cells, which possess p53 mutations, WT1 siRNA decreased WT1 protein to almost undetectable levels by 96-h post-transfection. Furthermore, WT1 siRNA transfection caused a significantly larger decrease in viability following irradiation than was seen in untransfected cells in both cell lines after treatment with ED50 of ionizing radiation. In conclusion, WT1 overexpression did not protect against p53-mediated apoptosis or ionizing radiation induced cell death. WT1 siRNA increased the radiosensitivity of two human glioma cell lines independently of p53. Anti-WT1 strategies may, therefore, prove useful in improving the response of glioblastoma to radiotherapy, thus potentially improving patient survival.

Tumor infiltration by myeloid suppressor cells in response to T cell activation in rat gliomas.

We have recently reported that activation of tumor-specific T cells by subcutaneous vaccination with irradiated T9 glioma cells of syngeneic rats with a pre-existing, intracranial (i.c.) T9 glioma (T9+vaccination) promotes the mobilization of myeloid suppressor cells (MSC) which inhibit T cell function resulting in unregulated tumor progression. The current study investigated if this immunological paradigm could be recapitulated in T cell deficient rats, in other rat glioma models or using a dendritic cell (DC) vaccine. When nude rats were used in the T9+vaccination model, the level of MSC tumor infiltration remained low in vaccinated and control groups and there was no significant difference in tumor size between the groups. Increased tumor infiltration by MSC after vaccination with respective irradiated tumor cells was observed in the 9L, F98 and D74 gliomas. RT-2 tumors were markedly infiltrated with MSC regardless of vaccination. Enhanced tumor progression in response to immunization and T cell activation was observed in rats bearing F98 and D74 gliomas, although less pronounced than in the T9 model, and there was a trend for increased tumor size in the 9L glioma model. Increased MSC infiltrate and augmented T9 glioma growth were observed when DC pulsed with T9 cell lysate was used as a vaccine. These results suggest that MSC infiltration and unregulated tumor growth in response to vaccination is T cell-dependent; is not unique to the T9 glioma; and can be recapitulated with an alternate immunization approach.

Irradiated tumor cell vaccine for treatment of an established glioma. I. Successful treatment with combined radiotherapy and cellular vaccination.

Rats bearing a 5-day intracranial (i.c.) syngeneic glioma were treated with a subcutaneous (s.c.) vaccination consisting of irradiated glioma cells or a multimodality approach composed of radiotherapy plus s.c. vaccination. Vaccination of rats harboring a T9 glioma with 5 x 10(6) irradiated T9.F glioma cells (a clone derived from the T9 glioblastoma cell line) resulted in a marked enhancement of i.c. glioma growth and a significant decrease in survival. Histopathology of the tumors from vaccinated rats revealed a massive glioma composed of healthy tumor tissue lacking any marked inflammation, edema or hemorrhage. Analysis of the tumor-infiltrating mononuclear cells indicated that gliomas from vaccinated rats contained a 10-fold greater lymphoid infiltrate per milligram of tumor as compared to tumors from non-vaccinated rats, suggesting that the vaccination had induced immune cells to localize to the i.c. glioma. Combined treatment consisting of 15 Gy of whole head irradiation of the 5-day glioma followed by vaccination with T9.F cells resulted in a significant increase in survival compared to that of non-treated rats, 45% of which remained tumor-free. Microscopic evaluation in survivors of the tumor implantation site revealed the presence of hemosiderin-laden macrophages and other mononuclear cells, with the absence of tumor cells within the residual lesion. When survivors were challenged s.c. with viable T9.F glioma cells, a delayed-type hypersensitivity (DTH) reaction appeared at the challenge site. T cells purified from these rats proliferated and secreted Th(1)-associated cytokines when stimulated with irradiated T9.F glioma cells, and lysed T9.F target cells. In contrast, when these rats were challenged s.c. with the unrelated MadB106 adenocarcinoma, tumor formation was observed. These findings indicate that the treatment of an established i.c. glioma with a cellular vaccination alone may induce enhanced tumor growth; however, when the vaccination is combined with radiation therapy, the results are beneficial in terms of increased survival time or complete remission that is accompanied by the development of tumor-specific cellular immunity.

Irradiated tumor cell vaccine for treatment of an established glioma. II. Expansion of myeloid suppressor cells that promote tumor progression.

These studies report the identification of a population of myeloid suppressor cells (MSC) that are preferentially enriched in the spleens and tumor-infiltrating mononuclear cells (TIMC) from T9.F-vaccinated animals. In this model designed to mimic immunotherapy for an established intracranial (i.c.) glioma, animals were given an i.c. inoculum with 5 x 10(4) T9 glioma cells at day 0, followed by a subcutaneous (s.c.) injection of 5 x 10(6) irradiated T9.F glioma cells 5 days later. Unexpectedly, we found that the survival of these T9.F-vaccinated animals was dramatically shorter than their age-matched counterparts who received only saline injections. Since MSC have previously been demonstrated to be associated with tumor progression, the question arose of whether MSC might play a role in the rapid tumor progression observed in this model. Analysis of the spleen cells and TIMC revealed an increase in the population of myeloid cells expressing granulocytic and monocytic markers. Both the polyclonal and tumor-specific proliferation of splenic T cells and tumor-infiltrating T lymphocytes (T-TIL) from T9.F-vaccinated animals were significantly inhibited in the presence of these myeloid cells. Furthermore, the adoptive transfer of MSC into animals bearing a 5-day T9 glioma caused rapid tumor progression. Reduced survival of the glioma-bearing vaccinated rats was associated with enhanced tumor growth, as well as with an increased density of T-TIL. However, purified T-TIL did not show any discernable signs of inherent defects in terms of their effector functions and T cell receptor (TCR) signal transduction protein levels. Therefore, we believe that an MSC population is responsible for inhibiting the anti-tumor T cell response, resulting in the enhanced growth of the i.c. glioma, and may represent a significant obstacle to immune-based therapies.

Thymic function and output of recent thymic emigrant T cells during intracranial glioma progression.

One of the hallmarks of patients with glioblastoma multiforme (GBM) is profound lymphopenia mostly confined to the T cell lineage. A deficiency in the production of naive T cells from the thymus could contribute to the lymphopenia seen in GBM patients. In this study we asked whether thymic function and the production of recent thymic emigrant (RTE) T cells from the thymus was influenced by intracranial (i.c.) glioma progression. We found significant thymic involution in animals with progressive i.c. gliomas. Involuted thymi from animals with progressive i.c. T9.F gliomas showed dramatic losses of CD4+ CD8+ (DP) thymocytes. Microscopic analysis complemented those findings by demonstrating a reversal of the typical cortico-medullary structure. Significant increases in apoptosis accompanied the rapid loss of viable thymocytes, which was prevented in part by adrenalectomy, suggesting a dominant role for endogenous glucocorticoids. This thymic involution was also associated with a significant decrease in peripheral RTE T cells, reflecting the diminished thymic function. Finally, we found that CD8+ RTE T cells were enriched in progressively growing T9 gliomas, which points to an immunological role for RTE's in anti-glioma immunity. Our findings may shed light on the significance of thymic function for anti-glioma immunity and the response to immunotherapeutic treatment paradigms.