NF-κB signaling plays a critical role in tumor growth and treatment resistance in GBM as in many other cancers. However, the molecular mechanisms underlying high, constitutive NF-κB activity in GBM remains to be elucidated. Here, we screened a panel of tripartite motif (TRIM) family proteins and identified TRIM22 as a potential activator of NF-κB using an NF-κB driven luciferase reporter construct in GBM cell lines. Knockout of TRIM22 using Cas9-sgRNAs led to reduced GBM cell proliferation, while TRIM22 overexpression enhanced proliferation of cell populations, in vitro and in an orthotopic xenograft model. However, two TRIM22 mutants, one with a critical RING-finger domain deletion and the other with amino acid changes at two active sites of RING E3 ligase (C15/18A), were both unable to promote GBM cell proliferation over controls, thus implicating E3 ligase activity in the growth-promoting properties of TRIM22. Co-immunoprecipitations demonstrated that TRIM22 bound a negative regulator of NF-κB, NF-κB inhibitor alpha (IκBα), and accelerated its degradation by inducing K48-linked ubiquitination. TRIM22 also formed a complex with the NF-κB upstream regulator IKKγ and promoted K63linked ubiquitination, which led to the phosphorylation of both IKKα/β and IκBα. Expression of a non-phosphorylation mutant, srIκBα, inhibited the growth-promoting properties of TRIM22 in GBM cell lines. Finally, TRIM22 was increased in a cohort of primary GBM samples on a tissue microarray, and high expression of TRIM22 correlated with other clinical parameters associated with progressive gliomas, such as wild-type IDH1 status. In summary, our study revealed that TRIM22 activated NF-κB signaling through posttranslational modification of two critical regulators of NF-κB signaling in GBM cells.
Recent evidence suggests that dysregulation of iron regulatory factors may play essential roles in cancer pathophysiology. Six-transmembrane epithelial antigen of prostate 3 (STEAP3) is a metalloreductase, which is vital for cellular iron uptake and homeostasis. However, the clinical significance and function of STEAP3 in the development of human gliomas remain unclear. Through analysis of publicly available databases, we found that STEAP3 was highly expressed in malignant gliomas, especially in the mesenchymal glioma molecular subtype and isocitrate dehydrogenase 1/2 (IDH1/2) wild-type gliomas. Expression levels of STEAP3 in gliomas correlated inversely with patient overall survival (OS) and served as an independent prognostic marker by multivariate Cox regression analysis. In functional assays performed with RNA knockdown, loss of STEAP3 attenuated aggressive phenotypes in glioma cells, including cell proliferation, invasion, and sphere formation in vitro and tumor growth in vivo. Finally, STEAP3 drives these activities by inducing mesenchymal transition, promoting transferrin receptor (TfR) expression, and activating STAT3-FoxM1 axis signaling. Taken together, these results indicate that STEAP3 functions as an oncogenic mediator in glioma progression and is thus a potential therapeutic target for the treatment of the disease.
Purpose: Long noncoding RNAs (lncRNA) have essential roles in diverse cellular processes, both in normal and diseased cell types, and thus have emerged as potential therapeutic targets. A specific member of this family, the SWI/SNF complex antagonist associated with prostate cancer 1 (SChLAP1), has been shown to promote aggressive prostate cancer growth by antagonizing the SWI/SNF complex and therefore serves as a biomarker for poor prognosis. Here, we investigated whether SChLAP1 plays a potential role in the development of human glioblastoma (GBM). Experimental Design: RNA-ISH and IHC were performed on a tissue microarray to assess expression of SChLAP1 and associated proteins in human gliomas. Proteins complexed with SChLAP1 were identified using RNA pull-down and mass spectrometry. Lentiviral constructs were used for functional analysis in vitro and in vivo. Results: SChLAP1 was increased in primary GBM samples and cell lines, and knockdown of the lncRNA suppressed growth. SChLAP1 was found to bind heterogeneous nuclear ribonucleoprotein L (HNRNPL), which stabilized the lncRNA and led to an enhanced interaction with the protein actinin alpha 4 (ACTN4). ACTN4 was also highly expressed in primary GBM samples and was associated with poorer overall survival in glioma patients. The SChLAP1–HNRNPL complex led to stabilization of ACTN4 through suppression of proteasomal degradation, which resulted in increased nuclear localization of the p65 subunit of NF-κB and activation of NF-κB signaling, a pathway associated with cancer development. Conclusions: Our results implicated SChLAP1 as a driver of GBM growth as well as a potential therapeutic target in treatment of the disease.
Increased Actin-like 6A (ACTL6A) expression has been implicated in the development of diverse cancers and recently associated with the Hippo signaling pathway, which is known to regulate biological properties, including proliferation, tissue regeneration, stem cell biology, as well as tumorigenesis. Here we first show that ACTL6A is upregulated in human gliomas and its expression is associated with glioma patient survival. ACTL6A promotes malignant behaviors of glioma cells in vitro and in orthotopic xenograft model. In co-immunoprecipitation assays, we discover that ACTL6A physically associated with YAP/TAZ and furthermore disrupts the interaction between YAP and β-TrCP E3 ubiquitin ligase, which promotes YAP protein degradation. Moreover, effects of ACTL6A on glioma cells proliferation, migration, and invasion could be mediated by YAP/TAZ. These data indicate that ACTL6A may contribute to cancer progression by stabilizing YAP/TAZ and therefore provide a novel therapeutic target for the treatment of human gliomas.
Increasing evidence demonstrates that ubiquitin specific protease 39 (USP39) plays an oncogenic role in various human tumors. Here, using expression analysis of the publicly available Oncomine database, clinical glioma patient samples, and glioma cells, we found that USP39 was overexpressed in human gliomas. Knockdown of USP39 in glioma cells demonstrated that the protein promoted cell growth, invasion and migration in vitro and in a tumor model in nude mice. To identify mediators of USP39 growth-promoting properties, we used luciferase reporter constructs under transcriptional control of various promoters specific to seven canonical cancer-associated pathways. Luciferase activity from a synthetic TEAD-dependent YAP/TAZ-responsive reporter, as a direct readout of the Hippo signaling pathway, was decreased by 92% in cells with USP39 knockdown, whereas the luciferase activities from the other six cancer pathways, including MAPK/ERK, MAPK/JNK, NFκB, Notch, TGFβ, and Wnt, remained unchanged. TAZ protein expression however was decreased independent of canonical Hippo signaling. Immunohistochemistry revealed a positive correlation between USP39 and TAZ proteins in orthotopic xenografts derived from modified glioma cells expressing USP39 shRNAs and primary human glioma samples (p < 0.05). Finally, loss of USP39 decreased TAZ pre-mRNA splicing efficiency in glioma cells in vitro, which led to reduced levels of TAZ protein. In summary, USP39 has oncogenic properties that increase TAZ protein levels by inducing maturation of its mRNA. USP39 therefore provides a novel therapeutic target for the treatment of human glioma.
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