Androgen-Regulated Transcriptional Control of Sialyltransferases in Prostate Cancer Cells

The expression of gangliosides is often associated with cancer progression. Sialyltransferases have received much attention in terms of their relationship with cancer because they modulate the expression of gangliosides. We previously demonstrated that GD1a production was high in castration-resistant prostate cancer cell lines, PC3 and DU145, mainly due to their high expression of β-galactoside α2,3-sialyltransferase (ST3Gal) II (not ST3Gal I), and the expression of both ST3Gals was regulated by NF-κB, mainly by RelB. We herein demonstrate that GD1a was produced in abundance in cancerous tissue samples from human patients with hormone-sensitive prostate cancers as well as castration-resistant prostate cancers. The expression of ST3Gal II was constitutively activated in castration-resistant prostate cancer cell lines, PC3 and DU145, because of the hypomethylation of CpG island in its promoter. However, in androgen-depleted LNCap cells, a hormone-sensitive prostate cancer cell line, the expression of ST3Gal II was silenced because of the hypermethylation of the promoter region. The expression of ST3Gal II in LNCap cells increased with testosterone treatment because of the demethylation of the CpG sites. This testosterone-dependent ST3Gal II expression was suppressed by RelB siRNA, indicating that RelB activated ST3Gal II transcription in the testosterone-induced demethylated promoter. Therefore, in hormone-sensitive prostate cancers, the production of GD1a may be regulated by androgen. This is the first report indicating that the expression of a sialyltransferase is transcriptionally regulated by androgen-dependent demethylation of the CpG sites in its gene promoter

Neuroblastoma chemotherapy can be augmented by immunotargeting O-acetyl-GD2 tumor-associated ganglioside

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Inhibition of tumor angiogenesis by globotriaosylceramide immunotargeting

International audienceCurrent antiangiogenic immunotherapeutic strategies mainly focus on the blockade of circulating cytokines or receptors that are overexpressed by endothelial cells. We proposed globotriaosylceramide (Gb3) as a viable alternative target for antiangiogenic therapies. In this setting, we developed an anti-Gb3 antibody and validated its therapeutic efficacy in metastatic tumor models. © 2013 Landes Bioscience

Intravenous injection of mAb c.8B6 does not induce allodynia.

<p>(A) Intraveinous injection of either 1 mg/kg (•) or 3 mg/kg (♦)of mAb c.8B6 in Sprague-Dawley rats did not result in a decrease in mechanical thresholds. In contrast, ch14.18 injected at 1 mg/kg (▪) resulted in a prolonged decrease in threshold. Threshold of animal injected with anti-CD20 chimeric control antibody rituximab (1 mg/kg) (□) are shown for comparison. (B) Results shown in panel B were recalculated as area under the curve and then normalized such that 100 would be equal to a theoretical maximal allodynia. Animals administered ch14.18 displayed more allodynia than any of the other treatment groups (p≤ 0.0001).</p

Chimeric Antibody c.8B6 to O-Acetyl-GD2 Mediates the Same Efficient Anti-Neuroblastoma Effects as Therapeutic ch14.18 Antibody to GD2 without Antibody Induced Allodynia

<div><p>Background</p><p>Anti-GD2 antibody is a proven therapy for GD2-postive neuroblastoma. Monoclonal antibodies against GD2, such as chimeric mAb ch14.18, have become benchmarks for neuroblastoma therapies. Pain, however, can limit immunotherapy with anti-GD2 therapeutic antibodies like ch14.18. This adverse effect is attributed to acute inflammation via complement activation on GD2-expressing nerves. Thus, new strategies are needed for the development of treatment intensification strategies to improve the outcome of these patients.</p><p>Methodology/Principal Findings</p><p>We established the mouse-human chimeric antibody c.8B6 specific to OAcGD2 in order to reduce potential immunogenicity in patients and to fill the need for a selective agent that can kill neuroblastoma cells without inducing adverse neurological side effects caused by anti-GD2 antibody immunotherapy. We further analyzed some of its functional properties compared with anti-GD2 ch14.18 therapeutic antibody. With the exception of allodynic activity, we found that antibody c.8B6 shares the same anti-neuroblastoma attributes as therapeutic ch14.18 anti-GD2 mAb when tested in cell-based assay and <i>in vivo</i> in an animal model.</p><p>Conclusion/Significance</p><p>The absence of OAcGD2 expression on nerve fibers and the lack of allodynic properties of c.8B6–which are believed to play a major role in mediating anti-GD2 mAb dose-limiting side effects–provide an important rationale for the clinical application of c.8B6 in patients with high-risk neuroblastoma.</p></div

Absence of mAb 8B6-specific OAcGD2 in mouse neuroblastoma sera.

<p>I-TLC of gangliosides from rat brain (Lane 0), NXS2 neuroblastoma tumor bearing mice sera (Lane 2), and mouse control sera (Lane 1) stained with resorcinol-HCl (Panel A) or immunostained with chimeric mAbs c.8B6 (Panel B), or ch14.18 (Panel C). Total ganglioside fractions presented in lanes 2 and 3 represents 1 ml of serum. Ganglioside GD2 was detected in the neuroblastoma-tumor bearing mice but not in the control sera when mAb ch14.18 was used. In contrast, no OAcGD2 was detected in sera from neuroblastoma-tumor bearing mice and control mouse sera when either mAb c.8B6 or ch14.18 was tested. Serums from 6 different mice were tested with the same results.</p

Immune effector functions of both mAbs c.8B6 and ch14.18.

<p>(A) Complement-dependent specific lysis was determined for the NXS2 cells as described in Materials and Methods. (B) The NK-92-RFcγIII+ ADCC activity with NXS2 target cells at E/T ratio 12 to 1 as described in Materials and Methods. Horizontally hatched columns, mAb c.8B6; white columns, mAb ch14.18; black columns, irrelevant antibody. Data are presented as the mean ± SEM for three independent experiments, each in triplicate.</p

Binding properties of mAbs c.8B6 and ch14.18 on NXS2 cells^*.

<p>*Determined as described in the Material and Methods Section. Data are presented as the mean ± SEM for three independent experiments, each in triplicate.</p

Anti-neuroblastoma activitiy of mAb c.8B6 against established experimental liver metastasis.

<p>Mice (n = 6/group) were inoculated with 1×10⁵ NXS2 cells by IV injections. Treatment was initiated three days after tumor cells inoculation and consisted of 5 daily IV injections of 25 µg of mAb c.8B6, mAb ch14.18, and anti-CD20 antibody. Mice were euthanised 28 days post-tumor cell inoculation. (A) The liver weight was determined on fresh specimen. The y-axis starts at 0.8 g corresponding to the average normal liver weight. Data are presented as the mean ± SEM. The differences in mean liver weight between experimental groups treated with mAbs c.8B6 and ch14.18 and all control groups (PBS, control antibody) was statistically significant (*p<0.05). (B) Representative liver specimen of each experimental group is shown. Arrows indicate the location of macroscopic liver metastasis. (Scale bar = 1 cm).</p

Binding specificity chimeric mAbs c.8B6 and ch14.18 measured by flow-cytometric analysis (A), by TLC-immunostaining (B) and by Immunochemistry on NXS2 tumor (C).

<p>(A) NXS2 and the OAcGD2/GD2⁻ Neuro2A cells were stained as described in Materials and Methods. Representative flow cytometry histogram of OAcGD2 expression. Antibody c.8B6 or mAb ch14.18 (<i>grey</i>) and control antibody (<i>black</i>). The same staining pattern was observed for mAb c.8B6 or ch14.18 on OAcGD2/GD2⁺ NXS2 cells whereas neither mAb c.8B6 nor mAb ch14.18 bind OAcGD2/GD2-negative Neuro-2a cells. These experiments were independently replicated 3 times. (B) TLC of gangliosides extracted from rat brain (Lane 0) and NXS2 cells with (Lane 1) or without (Lane 2) alkaline treatment and stained with resorcinol-HCl (Panel a) or immunostained with mAbs c.8B6 (Panel b), or ch14.18 (Panel c). Chimeric mAb 8B6 reacted with the alkali-labile <i>O</i>AcGD2 ganglioside with no cross-reactivity against GD2 ganglioside (Panel b) whereas ch14.18 reacted with GD2 and OAcGD2 (Panel c). The same results were obtained in 3 independent experiments. (C) An immunoperoxidase assay was performed on NX2 neuroblastoma tumor sections as described in Material and Methods. Strong immunostaining was detected on neuroblastoma cells with either mAb c.8B6 (c) or mAb ch14.18 (d). The anti-CD20 chimeric antibody ritximab was used as a negative control (b). No antibody (a). NXS2 neuroblastoma tumors from 6 different mice were tested with the same results. Scale bar = 20 µm.</p