Protein Stability Regulation in Osteosarcoma: The Ubiquitin-like Modifications and Glycosylation as Mediators of Tumor Growth and as Targets for Therapy.

The identification of new therapeutic targets and the development of innovative therapeutic approaches are the most important challenges for osteosarcoma treatment. In fact, despite being relatively rare, recurrence and metastatic potential, particularly to the lungs, make osteosarcoma a deadly form of cancer. In fact, although current treatments, including surgery and chemotherapy, have improved survival rates, the disease's recurrence and metastasis are still unresolved complications. Insights for analyzing the still unclear molecular mechanisms of osteosarcoma development, and for finding new therapeutic targets, may arise from the study of post-translational protein modifications. Indeed, they can influence and alter protein structure, stability and function, and cellular interactions. Among all the post-translational modifications, ubiquitin-like modifications (ubiquitination, deubiquitination, SUMOylation, and NEDDylation), as well as glycosylation, are the most important for regulating protein stability, which is frequently altered in cancers including osteosarcoma. This review summarizes the relevance of ubiquitin-like modifications and glycosylation in osteosarcoma progression, providing an overview of protein stability regulation, as well as highlighting the molecular mediators of these processes in the context of osteosarcoma and their possible targeting for much-needed novel therapy.

Anti-proliferative, pro-apototic and anti-migratory properties of HDAC inhibitor PXD-101 on osteosarcoma cell lines.

The therapeutic strategies for osteosarcoma involve both surgical approach and chemotherapy, but the identification of new therapeutic targets is particularly necessary in patients with local chemo-resistance, recurrence and lung metastases. The role of epigenetic regulation in osteosarcoma is largely unknown. Thus, in this study we disclosed the effects of histone deacetylase inhibitor drug PXD-101 on human osteosarcoma (OS) cell lines with different aggressiveness, including Saos-2, HOS and 143B cell lines. XTT assays revealed that treatment of Saos-2, HOS and 143B cells with PXD-101 decreased cell viability in a concentration-dependent manner. Fluorescence-activated cell sorting (FACS) analysis showed that PXD-101 inhibited proliferation and induced cell apoptosis. Wound healing assay indicated that PXD-101 inhibited migration of osteosarcoma cells. Real-Time RT-qPCR and protein analysis highlighted reduced expression of Runx2, Osterix and Mad2, probably due to Cyclin B1 inhibition by PXD-101 treatment. To our knowledge, this is the first study that characterized the anti-tumoral effect of PXD-101 in OS cells, suggesting a potential new therapeutic approach in osteosarcoma patients.

T-Cell Defects Associated to Lack of Spike-Specific Antibodies after BNT162b2 Full Immunization Followed by a Booster Dose in Patients with Common Variable Immune Deficiencies.

Following the third booster dose of the mRNA vaccine, Common Variable Immune Deficiencies (CVID) patients may not produce specific antibodies against the virus spike protein. The T-cell abnormalities associated with the absence of antibodies are still a matter of investigation. Spike-specific IgG and IgA, peripheral T cell subsets, CD40L and cytokine expression, and Spike-specific specific T-cells responses were evaluated in 47 CVID and 26 healthy donors after three doses of BNT162b2 vaccine. Testing was performed two weeks after the third vaccine dose. Thirty-six percent of the patients did not produce anti-SARS-CoV-2 IgG or IgA antibodies. Non responder patients had lower peripheral blood lymphocyte counts, circulating naïve and central memory T-cells, low CD40L expression on the CD4+CD45+RO+ and CD8+CD45+RO+ T-cells, high frequencies of TNFα and IFNγ expressing CD8+ T-cells, and defective release of IFNγ and TNFα following stimulation with Spike peptides. Non responders had a more complex disease phenotype, with higher frequencies of structural lung damage and autoimmunity, especially autoimmune cytopenia. Thirty-five percent of them developed a SARS-CoV-2 infection after immunization in comparison to twenty percent of CVID who responded to immunization with antibodies production. CVID-associated T cell abnormalities contributed to the absence of SARS-CoV-2 specific antibodies after full immunization.

Severe Acute Respiratory Syndrome Coronavirus 2 Monoclonal Antibody Combination Therapy in Patients With Coronavirus Disease 2019 and Primary Antibody Deficiency.

BACKGROUND: Previous reports highlighted the efficacy of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific monoclonal antibodies (mAbs) against coronavirus disease 2019. METHODS: We conducted a prospective study on the clinical outcome and antiviral effects of mAbs added to standard of care therapy in SARS-CoV-2-infected patients with primary antibody defects. RESULTS: Median time of SARS-CoV-2 quantitative polymerase chain reaction (qPCR) positivity was shorter in 8 patients treated with mAbs (22 days) than in 10 patients treated with standard of care therapy only (37 days, P=.026). Median time of SARS-CoV-2 qPCR positivity from mAb administration was 10 days. CONCLUSIONS: The SARS-CoV-2 mAbs treatment was effective and well tolerated in patients with primary antibody defects.

Subcellular Localization of uc.8+ as a Prognostic Biomarker in Bladder Cancer Tissue.

Non-coding RNA transcripts originating from Ultraconserved Regions (UCRs) have tissue-specific expression and play relevant roles in the pathophysiology of multiple cancer types. Among them, we recently identified and characterized the ultra-conserved-transcript-8+ (uc.8+), whose levels correlate with grading and staging of bladder cancer. Here, to validate uc.8+ as a potential biomarker in bladder cancer, we assessed its expression and subcellular localization by using tissue microarray on 73 human bladder cancer specimens. We quantified uc.8+ by in-situ hybridization and correlated its expression levels with clinical characteristics and patient survival. The analysis of subcellular localization indicated the simultaneous presence of uc.8+ in the cytoplasm and nucleus of cells from the Low-Grade group, whereas a prevalent cytoplasmic localization was observed in samples from the High-Grade group, supporting the hypothesis of uc.8+ nuclear-to-cytoplasmic translocation in most malignant tumor forms. Moreover, analysis of uc.8+ expression and subcellular localization in tumor-surrounding stroma revealed a marked down-regulation of uc.8+ levels compared to the paired (adjacent) tumor region. Finally, deep machine-learning approaches identified nucleotide sequences associated with uc.8+ localization in nucleus and/or cytoplasm, allowing to predict possible RNA binding proteins associated with uc.8+, recognizing also sequences involved in mRNA cytoplasm-translocation. Our model suggests uc.8+ subcellular localization as a potential prognostic biomarker for bladder cancer.

Author Correction: The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Role of PA2G4P4 pseudogene in bladder cancer tumorigenesis.

BACKGROUND: Many pseudogenes possess biological activities and play important roles in the pathogenesis of various types of cancer including bladder cancer (BlCa), which still lacks suitable molecular biomarkers. Recently, pseudogenes were found to be significantly enriched in a pan-cancer classification based on the Cancer Genome Atlas gene expression data. Among them, the top-ranking pseudogene was the proliferation-associated 2G4 pseudogene 4 (PA2G4P4). METHODS: Genomic and transcript features of PA2G4P4 were determined by GeneBank database analysis followed by 5' RACE experiments. Therefore, we conducted a retrospective molecular study on a cohort of 45 patients of BlCa. PA2G4P4 expression was measured by RT-qPCR, whereas PA2G4P4 transcript distribution was analyzed by in situ hybridization on both normal and cancerous histological sections and compared to the immunolocalization of its parental PA2G4/EBP1 protein. Finally, we tested the effects of PA2G4P4 depletion on proliferation, migration, and death of BlCa cells. RESULTS: We showed for the first time PA2G4P4 overexpression in BlCa tissues and in cell lines. PA2G4P4 distribution strictly overlaps PA2G4/EBP1 protein localization. Moreover, we showed that PA2G4P4 knockdown affects both proliferation and migration of BlCa cells, highlighting its potential oncogenic role. CONCLUSIONS: PA2G4P4 may play a functional role as an oncogene in BlCa development, suggesting it as a good candidate for future investigation and new clinical applications.

Publisher Correction: The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

Long non-coding RNAs (lncRNAs) are important regulatory molecules that are implicated in cellular physiology and pathology. In this work, we dissect the functional role of the HOXB-AS3 lncRNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 is shown to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism, HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts.

An Ultraconserved Element Containing lncRNA Preserves Transcriptional Dynamics and Maintains ESC Self-Renewal.

Ultraconserved elements (UCEs) show the peculiar feature to retain extended perfect sequence identity among human, mouse, and rat genomes. Most of them are transcribed and represent a new family of long non-coding RNAs (lncRNAs), the transcribed UCEs (T-UCEs). Despite their involvement in human cancer, the physiological role of T-UCEs is still unknown. Here, we identify a lncRNA containing the uc.170+, named T-UCstem1, and provide in vitro and in vivo evidence that it plays essential roles in embryonic stem cells (ESCs) by modulating cytoplasmic miRNA levels and preserving transcriptional dynamics. Specifically, while T-UCstem1::miR-9 cytoplasmic interplay regulates ESC proliferation by reducing miR-9 levels, nuclear T-UCstem1 maintains ESC self-renewal and transcriptional identity by stabilizing polycomb repressive complex 2 on bivalent domains. Altogether, our findings provide unprecedented evidence that T-UCEs regulate physiological cellular functions and point to an essential role of T-UCstem1 in preserving ESC identity.

The role of a new class of long noncoding RNAs transcribed from ultraconserved regions in cancer.

Ultraconserved regions (UCRs) represent a relatively new class of non-coding genomic sequences highly conserved between human, rat and mouse genomes. These regions can reside within exons of protein-coding genes, despite the vast majority of them localizes within introns or intergenic regions. Several studies have undoubtedly demonstrated that most of these regions are actively transcribed in normal cells/tissues, where they contribute to regulate many cellular processes. Interestingly, these non-coding RNAs exhibit aberrant expression levels in human cancer cells and their expression profiles have been used as prognostic factors in human malignancies, as well as to unambiguously distinguish among distinct cancer types. In this review, we first describe their identification, then we provide some updated information about their genomic localization and classification. More importantly, we discuss about the available literature describing an overview of the mechanisms through which some transcribed UCRs (T-UCR) contribute to cancer progression or to the metastatic spread. To date, the interplay between T-UCRs and microRNAs is the most convincing evidence linking T-UCRs and tumorigenesis. The limitations of these studies and the future challenges to be addressed in order to understand the biological role of T-UCRs are also discussed herein. We envision that future efforts are needed to convincingly include this class of ncRNAs in the growing area of cancer therapeutics.

Urinary long noncoding RNAs in nonmuscle-invasive bladder cancer: new architects in cancer prognostic biomarkers.

Several reports over the last 10 years provided evidence that long noncoding RNAs (lncRNAs) are often altered in bladder cancers. lncRNAs are longer than 200 nucleotides and function as important regulators of gene expression, interacting with the major pathways of cell growth, proliferation, differentiation, and survival. A large number of lncRNAs has oncogenic function and is more expressed in tumor compared with normal tissues. Their overexpression may be associated with tumor formation, progression, and metastasis in a variety of tumors including bladder cancer. Although lncRNAs have been shown to have critical regulatory roles in cancer biology, the biological functions and prognostic values in nonmuscle-invasive bladder cancer remain largely unknown. Nevertheless, a growing body of evidence suggests that several lncRNAs expression profiles in bladder malignancies are associated with poor prognosis, and they can be detected in biological fluids, such as urines. Here, we review current progress in the biology and the implication of lncRNAs associated with bladder cancer, and we discuss their potential use as diagnosis and prognosis biomarkers in bladder malignancies with a focus on their role in high-risk nonmuscle-invasive tumors.

New Cross-Talk Layer between Ultraconserved Non-Coding RNAs, MicroRNAs and Polycomb Protein YY1 in Bladder Cancer.

MicroRNAs (miRNAs) are highly conserved elements in mammals, and exert key regulatory functions. Growing evidence shows that miRNAs can interact with another class of non-coding RNAs, so-called transcribed ultraconserved regions (T-UCRs), which take part in transcriptional, post-transcriptional and epigenetic regulation processes. We report here the interaction of miRNAs and T-UCRs as a network modulating the availability of these non-coding RNAs in bladder cancer cells. In our cell system, antagomiR-596 increased the expression of T-UCR 201+. Moreover, T-UCR 8+ silencing increased miR-596 expression, which in turn reduced total T-UCR 283+, showing that the perturbation of one element in this network changes the expression of other interactors. In addition, we identify the polycomb protein Yin Yang 1 (YY1) as mediator of binding between miR-596 and T-UCR 8+. These new findings describe for the first time a network between T-UCRs, miRNAs and YY1 protein, highlighting the existence of an additional layer of gene expression regulation.

Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis.

Ultraconserved regions (UCRs) have been shown to originate non-coding RNA transcripts (T-UCRs) that have different expression profiles and play functional roles in the pathophysiology of multiple cancers. The relevance of these functions to the pathogenesis of bladder cancer (BlCa) is speculative. To elucidate this relevance, we first used genome-wide profiling to evaluate the expression of T-UCRs in BlCa tissues. Analysis of two datasets comprising normal bladder tissues and BlCa specimens with a custom T-UCR microarray identified ultraconserved RNA (uc.) 8+ as the most upregulated T-UCR in BlCa tissues, although its expression was lower than in pericancerous bladder tissues. These results were confirmed on BlCa tissues by real-time PCR and by in situ hybridization. Although uc.8+ is located within intron 1 of CASZ1, a zinc-finger transcription factor, the transcribed non-coding RNA encoding uc.8+ is expressed independently of CASZ1. In vitro experiments evaluating the effects of uc.8+ silencing, showed significantly decreased capacities for cancer cell invasion, migration, and proliferation. From this, we proposed and validated a model of interaction in which uc.8+ shuttles from the nucleus to the cytoplasm of BlCa cells, interacts with microRNA (miR)-596, and cooperates in the promotion and development of BlCa. Using computational analysis, we investigated the miR-binding domain accessibility, as determined by base-pairing interactions within the uc.8+ predicted secondary structure, RNA binding affinity, and RNA species abundance in bladder tissues and showed that uc.8+ is a natural decoy for miR-596. Thus uc.8+ upregulation results in increased expression of MMP9, increasing the invasive potential of BlCa cells. These interactions between evolutionarily conserved regions of DNA suggest that natural selection has preserved this potentially regulatory layer that uses RNA to modulate miR levels, opening up the possibility for development of useful markers for early diagnosis and prognosis as well as for development of new RNA-based cancer therapies.

Chemical modifications in the seed region of miRNAs 221/222 increase the silencing performances in gastrointestinal stromal tumor cells.

Most GastroIntestinal Stromal Tumors (GISTs) are characterized by KIT gene overexpression, which in turn is regulated by levels of microRNA 221 and microRNA 222. GISTs can also be distinguished by their miRNAs expression profile in which miRNAs 221/222 result reduced in comparison with GI normal tissues. In this paper, to restore normal miRNAs levels and to improve the silencing performances of miRNAs 221/222, new miRNA mimics in which guide strands are modified by Phosphorothioate (PS) and/or 2'-O-methyl RNA (2'-OMe) inside and outside the seed region, were synthesized and tested in GIST48 cells. We evaluated the positional effect of the chemical modifications on the miRNAs silencing activity, compared to natural and several commercial miRNA mimics. Our results show that chemically modified miRNAs 221/222 with alternating 2'-OMe-PS and natural nucleotides in the seed region are effective inhibitors of KIT gene expression and exhibit increased stability in rat plasma. Besides, their transfection in GIST 48 cells showed significant effects on different cellular processes in which KIT plays a functional role for tumor development (such as migration, cell proliferation, and apoptosis). Therefore, modified miRNAs 221/222 may provide an alternative therapeutic option for GIST treatment also aimed to overcome drug resistance concerns.