Retinoid receptor turnover mediated by sumoylation, ubiquitination and the valosin-containing protein is disrupted in glioblastoma.

Resistance to therapeutic use of retinoids in glioma has been observed for over 20 years; however, the exact mechanism of resistance remains unknown. To understand retinoic acid resistance in glioma, we studied the turnover mechanism of retinoid receptor proteins in neural stem cells and glioma stem-like cells. Here, we show that in normal neural stem cells, proteasomal degradation of retinoid receptors involves sumoylation, ubiquitination and recognition by the valosin-containing protein (VCP/p97/Cdc48). We find that Sumo1 modification has a dual role to stabilize the retinoid receptor from unwanted degradation and signal additional modification via ubiquitination. Subsequently, the modified receptor binds to the VCP chaperone and both proteins are degraded by the proteasome. Additionally, we reveal that all trans retinoic acid (ATRA) induces VCP expression, creating a positive feedback loop that enhances degradation. In contrast, the pathway is impaired in the glioma stem-like cells resulting in the accumulation of sumoylated and high molecular weight forms of retinoid receptors that lack transcriptional activity and fail to be recognized by the proteasome. Moreover, modified receptor accumulation occurs before ATRA treatment; therefore, the transcritptional defect in glioma is due to a block in the proteasomal degradation pathway that occurs after the sumo modification step.

Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy.

BACKGROUND: Corticosteroids are routinely utilized to alleviate edema in patients with intracranial lesions and are first-line agents to combat immune-related adverse events (irAEs) that arise with immune checkpoint blockade treatment. However, it is not known if or when corticosteroids can be administered without abrogating the efforts of immunotherapy. The purpose of this study was to evaluate the impact of dexamethasone on lymphocyte activation and proliferation during checkpoint blockade to provide guidance for corticosteroid use while immunotherapy is being implemented as a cancer treatment. METHODS: Lymphocyte proliferation, differentiation, and cytokine production were evaluated during dexamethasone exposure. Human T cells were stimulated through CD3 ligation and co-stimulated either directly by CD28 ligation or by providing CD80, a shared ligand for CD28 and CTLA-4. CTLA-4 signaling was inhibited by antibody blockade using ipilimumab which has been approved for the treatment of several solid tumors. The in vivo effects of dexamethasone during checkpoint blockade were evaluated using the GL261 syngeneic mouse intracranial model, and immune populations were profiled by flow cytometry. RESULTS: Dexamethasone upregulated CTLA-4 mRNA and protein in CD4 and CD8 T cells and blocked CD28-mediated cell cycle entry and differentiation. Naïve T cells were most sensitive, leading to a decrease of the development of more differentiated subsets. Resistance to dexamethasone was conferred by blocking CTLA-4 or providing strong CD28 co-stimulation prior to dexamethasone exposure. CTLA-4 blockade increased IFNγ expression, but not IL-2, in stimulated human peripheral blood T cells exposed to dexamethasone. Finally, we found that CTLA-4 blockade partially rescued T cell numbers in mice bearing intracranial gliomas. CTLA-4 blockade was associated with increased IFNγ-producing tumor-infiltrating T cells and extended survival of dexamethasone-treated mice. CONCLUSIONS: Dexamethasone-mediated T cell suppression diminishes naïve T cell proliferation and differentiation by attenuating the CD28 co-stimulatory pathway. However, CTLA-4, but not PD-1 blockade can partially prevent some of the inhibitory effects of dexamethasone on the immune response.

Identification of molecular pathways facilitating glioma cell invasion in situ.

Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion. In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC) xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs) compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT) processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.

Histone demethylase Jumonji D3 (JMJD3) as a tumor suppressor by regulating p53 protein nuclear stabilization.

Histone methylation regulates normal stem cell fate decisions through a coordinated interplay between histone methyltransferases and demethylases at lineage specific genes. Malignant transformation is associated with aberrant accumulation of repressive histone modifications, such as polycomb mediated histone 3 lysine 27 (H3K27me3) resulting in a histone methylation mediated block to differentiation. The relevance, however, of histone demethylases in cancer remains less clear. We report that JMJD3, a H3K27me3 demethylase, is induced during differentiation of glioblastoma stem cells (GSCs), where it promotes a differentiation-like phenotype via chromatin dependent (INK4A/ARF locus activation) and chromatin independent (nuclear p53 protein stabilization) mechanisms. Our findings indicate that deregulation of JMJD3 may contribute to gliomagenesis via inhibition of the p53 pathway resulting in a block to terminal differentiation.

Glycogen synthase kinase-3 inhibition induces glioma cell death through c-MYC, nuclear factor-kappaB, and glucose regulation.

Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, is involved in diverse cellular processes ranging from nutrient and energy homeostasis to proliferation and apoptosis. Its role in glioblastoma multiforme has yet to be elucidated. We identified GSK3 as a regulator of glioblastoma multiforme cell survival using microarray analysis and small-molecule and genetic inhibitors of GSK3 activity. Various molecular and genetic approaches were then used to dissect out the molecular mechanisms responsible for GSK3 inhibition-induced cytotoxicity. We show that multiple small molecular inhibitors of GSK3 activity and genetic down-regulation of GSK3alpha/beta significantly inhibit glioma cell survival and clonogenicity. The potency of the cytotoxic effects is directly correlated with decreased enzyme activity-activating phosphorylation of GSK3alpha/beta Y276/Y216 and with increased enzyme activity inhibitory phosphorylation of GSK3alpha S21. Inhibition of GSK3 activity results in c-MYC activation, leading to the induction of Bax, Bim, DR4/DR5, and tumor necrosis factor-related apoptosis-inducing ligand expression and subsequent cytotoxicity. Additionally, down-regulation of GSK3 activity results in alteration of intracellular glucose metabolism resulting in dissociation of hexokinase II from the outer mitochondrial membrane with subsequent mitochondrial destabilization. Finally, inhibition of GSK3 activity causes a dramatic decrease in intracellular nuclear factor-kappaB activity. Inhibition of GSK3 activity results in c-MYC-dependent glioma cell death through multiple mechanisms, all of which converge on the apoptotic pathways. GSK3 may therefore be an important therapeutic target for gliomas. Future studies will further define the optimal combinations of GSK3 inhibitors and cytotoxic agents for use in gliomas and other cancers.