GRP78 determines glioblastoma sensitivity to UBA1 inhibition-induced UPR signaling and cell death

Glioblastoma multiforme (GBM) is an extremely aggressive brain tumor for which new therapeutic approaches are urgently required. Unfolded protein response (UPR) plays an important role in the progression of GBM and is a promising target for developing novel therapeutic interventions. We identified ubiquitin-activating enzyme 1 (UBA1) inhibitor TAK-243 that can strongly induce UPR in GBM cells. In this study, we evaluated the functional activity and mechanism of TAK-243 in preclinical models of GBM. TAK-243 significantly inhibited the survival, proliferation, and colony formation of GBM cell lines and primary GBM cells. It also revealed a significant anti-tumor effect on a GBM PDX animal model and prolonged the survival time of tumor-bearing mice. Notably, TAK-243 more effectively inhibited the survival and self-renewal ability of glioblastoma stem cells (GSCs) than GBM cells. Importantly, we found that the expression level of GRP78 is a key factor in determining the sensitivity of differentiated GBM cells or GSCs to TAK-243. Mechanistically, UBA1 inhibition disrupts global protein ubiquitination in GBM cells, thereby inducing ER stress and UPR. UPR activates the PERK/ATF4 and IRE1α/XBP signaling axes. These findings indicate that UBA1 inhibition could be an attractive strategy that may be potentially used in the treatment of patients with GBM, and GRP78 can be used as a molecular marker for personalized treatment by targeting UBA1.Subject terms: CNS cancer, Cancer stem cells     

Multi-Kinase Inhibitor C1 Triggers Mitotic Catastrophe of Glioma Stem Cells Mainly through MELK Kinase Inhibition

Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.     

Rosuvastatin reduces microglia in the brain of wild type and ApoE knockout mice on a high cholesterol diet; implications for prevention of stroke and AD

IGFBP2 is overexpressed in the most common brain tumor, glioblastoma (GBM), and its expression is inversely correlated to GBM patient survival. Previous reports have demonstrated a role for IGFBP2 in glioma cell invasion and astrocytoma development. However, the function of IGFBP2 in the restricted, self-renewing, and tumorigenic GBM cell population comprised of tumor-initiating stem cells has yet to be determined. Herein we demonstrate that IGFBP2 is overexpressed within the stem cell compartment of GBMs and is integral for the clonal expansion and proliferative properties of glioma stem cells (GSCs). In addition, IGFBP2 inhibition reduced Akt-dependent GSC genotoxic and drug resistance. These results suggest that IGFBP2 is a selective malignant factor that may contribute significantly to GBM pathogenesis by enriching for GSCs and mediating their survival. Given the current dearth of selective molecular targets against GSCs, we anticipate our results to be of high therapeutic relevance in combating the rapid and lethal course of GBM.     

The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.     

Elevated invasive potential of glioblastoma stem cells

Glioblastomas (GBMs) are the most lethal and common types of primary brain tumors. The hallmark of GBMs is their highly infiltrative nature. The cellular and molecular mechanisms underlying the aggressive cancer invasion in GBMs are poorly understood. GBM displays remarkable cellular heterogeneity and hierarchy containing self-renewing glioblastoma stem cells (GSCs). Whether GSCs are more invasive than non-stem tumor cells and contribute to the invasive phenotype in GBMs has not been determined. Here we provide experimental evidence supporting that GSCs derived from GBM surgical specimens or xenografts display greater invasive potential in vitro and in vivo than matched non-stem tumor cells. Furthermore, we identified several invasion-associated proteins that were differentially expressed in GSCs relative to non-stem tumor cells. One of such proteins is L1CAM, a cell surface molecule shown to be critical to maintain GSC tumorigenic potential in our previous study. Immunohistochemical staining showed that L1CAM is highly expressed in a population of cancer cells in the invasive fronts of primary GBMs. Collectively, these data demonstrate the invasive nature of GSCs, suggesting that disrupting GSCs through a specific target such as L1CAM may reduce GBM cancer invasion and tumor recurrence.     

Deubiquitinase USP39 and E3 ligase TRIM26 balance the level of ZEB1 ubiquitination and thereby determine the progression of hepatocellular carcinoma

Emerging evidence suggests that USP39 plays an important role in the development of hepatocellular carcinoma (HCC). However, the molecular mechanism by which USP39 promotes HCC progression has not been well defined, especially regarding its putative ubiquitination function. Zinc-finger E-box-binding homeobox 1 (ZEB1) is a crucial inducer of epithelial-to-mesenchymal transition (EMT) to promote tumor proliferation and metastasis, but the regulatory mechanism of ZEB1 stability in HCC remains enigmatic. Here, we reveal that USP39 is highly expressed in human HCC tissues and correlated with poor prognosis. Moreover, USP39 depletion inhibits HCC cell proliferation and metastasis by promoting ZEB1 degradation. Intriguingly, deubiquitinase USP39 has a direct interaction with the E3 ligase TRIM26 identified by co-immunoprecipitation assays and immunofluorescence staining assays. We further demonstrate that TRIM26 is lowly expressed in human HCC tissues and inhibits HCC cell proliferation and migration. TRIM26 promotes the degradation of ZEB1 protein by ubiquitination in HCC. Deubiquitinase USP39 and E3 ligase TRIM26 function in an antagonistic pattern, but not a competitive pattern, and play key roles in controlling ZEB1 stability to determine the HCC progression. In summary, our data reveal a previously unknown mechanism that USP39 and TRIM26 balance the level of ZEB1 ubiquitination and thereby determine HCC cell proliferation and migration. This novel mechanism may provide new approaches to target treatment for inhibiting HCC development by restoring TRIM26 or suppressing USP39 expression in HCC cases with high ZEB1 protein levels.Subject terms: Gene regulation, Prognostic markers     

Serum-Induced Differentiation of Glioblastoma Neurospheres Leads to Enhanced Migration/Invasion Capacity That Is Associated with Increased MMP9

Glioblastoma (GBM) is a highly infiltrative brain tumor in which cells with properties of stem cells, called glioblastoma stem cells (GSCs), have been identified. In general, the dominant view is that GSCs are responsible for the initiation, progression, invasion and recurrence of this tumor. In this study, we addressed the question whether the differentiation status of GBM cells is associated with their invasive capacity. For this, several primary GBM cell lines were used, cultured either as neurospheres known to enrich for GSCs or in medium supplemented with 10% FCS that promotes differentiation. The differentiation state of the cells was confirmed by determining the expression of stem cell and differentiation markers. The migration/invasion potential of these cells was tested using in vitro assays and intracranial mouse models. Interestingly, we found that serum-induced differentiation enhanced the invasive potential of GBM cells, which was associated with enhanced MMP9 expression. Chemical inhibition of MMP9 significantly reduced the invasive potential of differentiated cells in vitro. Furthermore, the serum-differentiated cells could revert back to an undifferentiated/stem cell state that were able to form neurospheres, although with a reduced efficiency as compared to non-differentiated counterparts. We propose a model in which activation of the differentiation program in GBM cells enhances their infiltrative potential and that depending on microenvironmental cues a significant portion of these cells are able to revert back to an undifferentiated state with enhanced tumorigenic potential. Thus, effective therapy should target both GSCs and differentiated offspring and targeting of differentiation-associated pathways may offer therapeutic opportunities to reduce invasive growth of GBM.     

Overexpression of FOXD2‐AS1 enhances proliferation and impairs differentiation of glioma stem cells by activating the NOTCH pathway via TAF‐1

Emerging data have highlighted the importance of long noncoding RNAs (lncRNAs) in exerting critical biological functions and roles in different forms of brain cancer, including gliomas. In this study, we sought to investigate the role of lncRNA FOXD2 adjacent opposite strand RNA 1 (FOXD2‐AS1) in glioma cells. First, we used sphere formation assay and flow cytometry to select U251 glioma stem cells (GSCs). Then, we quantified the expression of lncRNA FOXD2‐AS1, TATA‐box binding protein associated factor 1 (TAF‐1) and NOTCH1 in glioma tissues and GSCs, as well as the expression of GSC stem markers, OCT4, SOX2, Nanog, Nestin and CD133 in GSCs. Colony formation assay, sphere formation assay, and flow cytometry were used to evaluate GSC stemness. Next, the correlations among lncRNA FOXD2‐AS1, TAF‐1 and NOTCH1 were investigated. LncRNA FOXD2‐AS1, TAF‐1 and NOTCH1 were found to be elevated in glioma tissues and GSCs, and silencing lncRNA FOXD2‐AS1 inhibited stemness and proliferation, while promoting apoptosis and differentiation of GSCs. LncRNA FOXD2‐AS1 overexpression also led to increased NOTCH1 by recruiting TAF‐1 to the NOTCH1 promoter region, thereby promoting stemness and proliferation, while impairing cell apoptosis and differentiation. Mechanistically, lncRNA FOXD2‐AS1 elevation promoted glioma in vivo by activating the NOTCH signalling pathway via TAF‐1 upregulation. Taken together, the key findings of our investigation support the proposition that downregulation of lncRNA FOXD2‐AS1 presents a viable and novel molecular candidate for improving glioma treatment.     

L1CAM regulates DNA damage checkpoint response of glioblastoma stem cells through NBS1

Glioblastomas (GBMs) are highly lethal brain tumours with current therapies limited to palliation due to therapeutic resistance. We previously demonstrated that GBM stem cells (GSCs) display a preferential activation of DNA damage checkpoint and are relatively resistant to radiation. However, the molecular mechanisms underlying the preferential checkpoint response in GSCs remain undefined. Here, we show that L1CAM (CD171) regulates DNA damage checkpoint responses and radiosensitivity of GSCs through nuclear translocation of L1CAM intracellular domain (L1-ICD). Targeting L1CAM by RNA interference attenuated DNA damage checkpoint activation and repair, and sensitized GSCs to radiation. L1CAM regulates expression of NBS1, a critical component of the MRE11-RAD50-NBS1 (MRN) complex that activates ataxia telangiectasia mutated (ATM) kinase and early checkpoint response. Ectopic expression of NBS1 in GSCs rescued the decreased checkpoint activation and radioresistance caused by L1CAM knockdown, demonstrating that L1CAM signals through NBS1 to regulate DNA damage checkpoint responses. Mechanistically, nuclear translocation of L1-ICD mediates NBS1 upregulation via c-Myc. These data demonstrate that L1CAM augments DNA damage checkpoint activation and radioresistance of GSCs through L1-ICD-mediated NBS1 upregulation and the enhanced MRN-ATM-Chk2 signalling.     

Ubiquitin-specific peptidase 8 regulates the trafficking and stability of the human organic anion transporter 1

BackgroundOrganic anion transporter 1 (OAT1) plays a vital role in avoiding the potential toxicity of various anionic drugs through the involvement of kidney elimination. We previously demonstrated that ubiquitin conjugation to OAT1 led to OAT1 internalization from cell surface, followed by degradation. Ubiquitination is a dynamic process, where deubiquitination is catalyzed by a class of ubiquitin-specific peptidases.MethodsThe role of ubiquitin-specific peptidase 8 (USP8) in hOAT1 function, expression and ubiquitination was assessed by conducting transporter uptake assay, biotinylation assay and ubiquitination assay.ResultsWe demonstrated that USP8 overexpression in hOAT1-expressing cells led to an increased hOAT1 transporter activity and expression, which correlated well with a reduced hOAT1 ubiquitination. Such phenomenon was not observed in inactive USP8 mutant-transfected cells. In addition, the knockdown of endogenous USP8 by USP8-specific siRNA resulted in an increased hOAT1 ubiquitination, which correlated well with a decrease in hOAT1 expression and transport activity. Biotinylation experiments demonstrated that USP8-induced increase in hOAT1 expression and transport activity occurred through a deceleration of the rates of hOAT1 internalization and degradation.ConclusionsThese results indicated the regulatory role of USP8 in OAT1 function, expression, trafficking, and stability.General significanceUSP8 could be a new target for modulating OAT1-mediated drug transport.     

Cancer stem cells in glioblastoma — molecular signaling and therapeutic targeting

Glioblastomas (GBMs) are highly lethal primary brain tumors. Despite current therapeutic advances in other solid cancers, the treatment of these malignant gliomas remains essentially palliative. GBMs are extremely resistant to conventional radiation and chemotherapies. We and others have demonstrated that a highly tumorigenic subpopulation of cancer cells called GBM stem cells (GSCs) promotes therapeutic resistance. We also found that GSCs stimulate tumor angiogenesis by expressing elevated levels of VEGF and contribute to tumor growth, which has been translated into a useful therapeutic strategy in the treatment of recurrent or progressive GBMs. Furthermore, stem cell-like cancer cells (cancer stem cells) have been shown to promote metastasis. Although GBMs rarely metastasize beyond the central nervous system, these highly infiltrative cancers often invade into normal brain tissues preventing surgical resection, and GSCs display an aggressive invasive phenotype. These studies suggest that targeting GSCs may effectively reduce tumor recurrence and significantly improve GBM treatment. Recent studies indicate that cancer stem cells share core signaling pathways with normal somatic or embryonic stem cells, but also display critical distinctions that provide important clues into useful therapeutic targets. In this review, we summarize the current understanding and advances in glioma stem cell research, and discuss potential targeting strategies for future development of anti-GSC therapies.     

Regulation of Epidermal Growth Factor Receptor Ubiquitination and Trafficking by the USP8·STAM Complex

Reversible ubiquitination of activated receptor complexes signals their sorting between recycling and degradation and thereby dictates receptor fate. The deubiquitinating enzyme ubiquitin-specific protease 8 (USP8/UBPy) has been previously implicated in the regulation of the epidermal growth factor receptor (EGFR); however, the molecular mechanisms governing its recruitment and activity in this context remain unclear. Herein, we investigate the role of USP8 in countering ligand-induced ubiquitination and down-regulation of EGFR and characterize a subset of protein-protein interaction determinants critical for this function. USP8 depletion accelerates receptor turnover, whereas loss of hepatocyte growth factor-regulated substrate (Hrs) rescues this phenotype, indicating that USP8 protects EGFR from degradation via an Hrs-dependent pathway. Catalytic inactivation of USP8 incurs EGFR hyperubiquitination and promotes receptor localization to endosomes marked by high ubiquitin content. These phenotypes require the central region of USP8, containing three extended Arg-X-X-Lys (RXXK) motifs that specify direct low affinity interactions with the SH3 domain(s) of ESCRT-0 proteins, STAM1/2. The USP8·STAM complex critically impinges on receptor ubiquitination status and modulates ubiquitin dynamics on EGFR-positive endosomes. Consequently, USP8-mediated deubiquitination slows progression of EGFR past the early-to-recycling endosome circuit in a manner dependent upon the RXXK motifs. Collectively, these findings demonstrate a role for the USP8·STAM complex as a protective mechanism regulating early endosomal sorting of EGFR between pathways destined for lysosomal degradation and recycling.