Glycoprotein nonmetastatic melanoma protein B impacts the malignant potential of bladder cancer cells through its hem-immunoreceptor tyrosine-based activation motif.

Bladder cancer is one of the most common cancers among men worldwide. Although multiple genomic mutations and epigenetic alterations have been identified, an efficacious molecularly targeted therapy has yet to be established. Therefore, a novel approach is anticipated. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein that is highly expressed in various cancers. In this study, we evaluated bladder cancer patient samples and found that GPNMB protein abundance is associated with high-grade tumors, and both univariate and multivariate analyses showed that GPNMB is a prognostic factor. Furthermore, the prognosis of patients with high GPNMB levels was significantly poorer in those with nonmuscle invasive bladder cancer (NMIBC) than in those with muscle invasive bladder cancer (MIBC). We then demonstrated that knockdown of GPNMB in MIBC cell lines with high GPNMB inhibits cellular migration and invasion, whereas overexpression of GPNMB further enhances cellular migration and invasion in MIBC cell lines with originally low GPNMB. Therefore, we propose that GPNMB is one of multiple driver molecules in the acquisition of cellular migratory and invasive potential in bladder cancers. Moreover, we revealed that the tyrosine residue in the hemi-immunoreceptor tyrosine-based activation motif (hemITAM) is required for GPNMB-induced cellular motility.

Glycoprotein non-metastatic melanoma protein B functions with growth factor signaling to induce tumorigenesis through its serine phosphorylation.

Breast cancer is the most common cancer among women. Glycoprotein non-metastatic melanoma protein B (GPNMB), a type I transmembrane protein that is highly expressed in many cancers, including breast cancer, has been shown to be a prognostic factor. We previously reported that GPNMB overexpression confers tumorigenic potential, as evidenced by invasive tumor growth in vivo, sphere formation, and cellular migration and invasion to non-tumorigenic mammary epithelial cells. In this study, we focused on the serine (S) residue in the intracellular domain of GPNMB (S530 in human isoform b and S546 in mouse), which is predicted to be a phosphorylation site. To investigate the roles of this serine residue, we made an antibody specific for S530-phosphorylated human GPNMB and a point mutant in which S530 is replaced by an alanine (A) residue, GPNMB(SA). Established GPNMB(SA) overexpressing cells showed a significant reduction in sphere formation in vitro and tumor growth in vivo as a result of decreased stemness-related gene expression compared to that in GPNMB(WT)-expressing cells. In addition, GPNMB(SA) impaired GPNMB-mediated cellular migration. Furthermore, we found that tyrosine kinase receptor signaling triggered by epidermal growth factor or fibroblast growth factor 2 induces the serine phosphorylation of GPNMB through activation of downstream oncoproteins RAS and RAF.

Sustained release of PKR inhibitor C16 from mesoporous silica nanoparticles significantly enhances mRNA translation and anti-tumor vaccination.

In this study, we developed a mesoporous silica nanoparticles - mRNA (MSN-mRNA) subcutaneous delivery system composed of naked mRNA and a subcutaneous depot of imidazolo-oxindole RNA-activated protein kinase (PKR) inhibitor C16. We show that C16 treatment during mRNA transfection is a potent immune evasion approach that non-linearly enhances translation of unmodified mRNA in both mouse fibroblasts and dendritic cells in vitro exceeding that of nucleoside-modified mRNA. Notably, C16 further enhances translation of nucleoside-modified mRNA and HPLC purified mRNA. However, translation enhancement is dependent on and potentiated by C16's continuous presence. C16 mediated translation enhancement is extended in vivo by employing MSN as an interface to sustain-release C16. Subcutaneously administered MSN-mRNA significantly enhanced in vivo translation and expression kinetics of naked mRNA in unmodified, nucleoside-modified, and HPLC purified formats. We applied a MSN-mRNA vaccine formulation composed of naked mRNA encoding ovalbumin and granulocyte macrophage colony stimulating factor, and C16@MSNs on a xenograft E.G7-OVA prophylactic tumor model, resulting in very potent tumor inhibition. The MSN-mRNA delivery system bears great translational potential in mRNA therapeutics.

Messenger RNA translation enhancement by immune evasion proteins: a comparative study between EKB (vaccinia virus) and NS1 (influenza A virus).

In this study, we compared vaccinia virus derived monofunctional E3, K3 and B18R (also known as EKB) with influenza A virus derived multifunctional non-structural protein 1 (NS1) based on their ability to enhance mRNA translation. EKB and NS1-TX91 were all found to enhance mRNA translation and suppress interferon production, yet level of enhancement by EKB was much lower than NS1-TX91. Similarly, greater luciferase expression was mediated by co-delivery of unmodified luciferase with NS1 mRNA, compared to co-delivery of unmodified luciferase with either E3, K3 or B18R mRNA, respectively. Different combinations of E3, K3 and/or B18R mRNA were mixed with NS1-TX91 mRNA at varying ratios and co-delivered with luciferase mRNA. However, no synergism was observed as mRNA translation enhancement mediated by NS1-TX91 could not be improved by the inclusion EKB in all tested combinations. Lastly, it was found that E3 was able to rescue mRNA translation enhancement mediated by NS1 PKR knockout mutant (PR8PKR-), suggesting that one of NS1's multiple immune evasion mechanisms overlapped with E3. Altogether, our data validated mRNA translation enhancement mediated by immune evasion proteins (EKB and NS1) and showed that the multifunctional nature of NS1 accounted for its superior performance.

Unravelling the RNA-Binding Properties of SAFB Proteins in Breast Cancer Cells.

Scaffold attachment factor B1 (SAFB1) and SAFB2 proteins are oestrogen (ER) corepressors that bind to and modulate ER activity through chromatin remodelling or interaction with the basal transcription machinery. SAFB proteins also have an internal RNA-recognition motif but little is known about the RNA-binding properties of SAFB1 or SAFB2. We utilised crosslinking and immunoprecipitation (iCLIP) coupled with high-throughput sequencing to enable a transcriptome-wide mapping of SAFB1 protein-RNA interactions in breast cancer MCF-7 cells. Analysis of crosslinking frequency mapped to transcript regions revealed that SAFB1 binds to coding and noncoding RNAs (ncRNAs). The highest proportion of SAFB1 crosslink sites mapped to ncRNAs, followed by intergenic regions, open reading frames (ORFs), introns, and 3' or 5' untranslated regions (UTR). Furthermore, we reveal that SAFB1 binds directly to RNA and its binding is particularly enriched at purine-rich sequences not dissimilar to the RNA-binding motifs for SR proteins. Using RNAi, we also show, for the first time, that single depletion of either SAFB1 or SAFB2 leads to an increase in expression of the other SAFB protein in both MCF-7 and MDA-MD231 breast cancer cells.