E3 Ubiquitin Ligase TRIM Proteins, Cell Cycle and Mitosis

The cell cycle is a series of events by which cellular components are accurately segregated into daughter cells, principally controlled by the oscillating activities of cyclin-dependent kinases (CDKs) and their co-activators. In eukaryotes, DNA replication is confined to a discrete synthesis phase while chromosome segregation occurs during mitosis. During mitosis, the chromosomes are pulled into each of the two daughter cells by the coordination of spindle microtubules, kinetochores, centromeres, and chromatin. These four functional units tie chromosomes to the microtubules, send signals to the cells when the attachment is completed and the division can proceed, and withstand the force generated by pulling the chromosomes to either daughter cell. Protein ubiquitination is a post-translational modification that plays a central role in cellular homeostasis. E3 ubiquitin ligases mediate the transfer of ubiquitin to substrate proteins determining their fate. One of the largest subfamilies of E3 ubiquitin ligases is the family of the tripartite motif (TRIM) proteins, whose dysregulation is associated with a variety of cellular processes and directly involved in human diseases and cancer. In this review we summarize the current knowledge and emerging concepts about TRIMs and their contribution to the correct regulation of cell cycle, describing how TRIMs control the cell cycle transition phases and their involvement in the different functional units of the mitotic process, along with implications in cancer progression

circPVT1 and PVT1/AKT3 show a role in cell proliferation, apoptosis, and tumor subtype-definition in small cell lung cancer

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.</p

E3 Ubiquitin Ligase TRIM Proteins, Cell Cycle and Mitosis

The cell cycle is a series of events by which cellular components are accurately segregated into daughter cells, principally controlled by the oscillating activities of cyclin-dependent kinases (CDKs) and their co-activators. In eukaryotes, DNA replication is confined to a discrete synthesis phase while chromosome segregation occurs during mitosis. During mitosis, the chromosomes are pulled into each of the two daughter cells by the coordination of spindle microtubules, kinetochores, centromeres, and chromatin. These four functional units tie chromosomes to the microtubules, send signals to the cells when the attachment is completed and the division can proceed, and withstand the force generated by pulling the chromosomes to either daughter cell. Protein ubiquitination is a post-translational modification that plays a central role in cellular homeostasis. E3 ubiquitin ligases mediate the transfer of ubiquitin to substrate proteins determining their fate. One of the largest subfamilies of E3 ubiquitin ligases is the family of the tripartite motif (TRIM) proteins, whose dysregulation is associated with a variety of cellular processes and directly involved in human diseases and cancer. In this review we summarize the current knowledge and emerging concepts about TRIMs and their contribution to the correct regulation of cell cycle, describing how TRIMs control the cell cycle transition phases and their involvement in the different functional units of the mitotic process, along with implications in cancer progression

Dissecting the TRIM8 role in the pathogenesis of glioma

I gliomi umani sono un gruppo eterogeneo di tumori cerebrali maligni primari, la cui patogenesi molecolare risulta ancora parzialmente sconosciuta. Pertanto, la comprensione dei meccanismi molecolari alla base della loro insorgenza e del loro decorso può portare a una migliore scelta di terapie appropriate e a migliori risultati prognostici, attraverso l'identificazione di nuovi geni specifici associati al glioma. Le proteine appartenenti alla famiglia “tripartite motif” (TRIM) sono coinvolte in diversi processi biologici, tra cui la regolazione trascrizionale, il controllo della progressione del ciclo cellulare, la proliferazione e il differenziamento cellulare. Alterazioni dell’espressione delle proteine TRIM sono associate a una varietà di patologie quali disturbi dello sviluppo, malattie infiammatorie e tumori. Tra le circa 80 proteine identificate appartenenti alla famiglia delle TRIM, TRIM8 è una E3 ubiquitina-ligasi coinvolta in vari processi patologici, quali ipertrofia, risposta antivirale, encefalopatia e sviluppo di diverse forme di cancro. Abbiamo recentemente identificato TRIM8 come un gene differenzialmente espresso nei gliomi, la cui espressione è correlata a un esito clinico sfavorevole nei pazienti con glioma. Per ottenere informazioni approfondite sulle funzioni di TRIM8, ne abbiamo studiato il “trascrittoma” e l' “interattoma” in cellule staminali neurali embrionali di topo, usando l’RNA-Sequencing e la spettrometria di massa, e successivamente analizzando i dati ottenuti mediante programmi bioinformatici. Sono state quindi eseguite analisi funzionali, biochimiche e cellulari, per esplorare il ruolo TRIM8 in differenti processi biologici. Il nostro studio ci ha permesso di identificare vie di segnale correlate alla neurotrasmissione e al sistema nervoso centrale (SNC), fornendo ulteriori prove dell'esistenza di una relazione funzionale tra TRIM8 e STAT3, con possibili implicazioni nello sviluppo e nella progressione del glioma. Abbiamo successivamente dimostrato che TRIM8 interagisce con KIFC1 e KIF11/Eg5, due importanti regolatori dell'assemblaggio del fuso mitotico e della riorganizzazione del citoscheletro. Approfondendo lo studio sul ruolo di TRIM8 nel processo mitotico, abbiamo verificato che TRIM8 localizza a livello del fuso mitotico durante la progressione della mitosi e svolge un ruolo chiave nella separazione dei centrosomi all’inizio della divisione mitotica, con un conseguente ritardo nella progressione della mitosi e un impatto sulla stabilità cromosomica. I nostri risultati confermano il ruolo di TRIM8 nelle funzioni cerebrali attraverso la deregolazione di geni appartenenti al pathway JAK-STAT e coinvolti in diverse funzioni del SNC. Inoltre, abbiamo identificato la funzione fisiologica di TRIM8 nello sviluppo del fuso mitotico, evidenziando un ruolo emergente di TRIM8 nella regolazione della mitosi.Human gliomas are a heterogeneous group of primary malignant brain tumors, whose molecular pathogenesis is not yet solved. Therefore, understanding the molecular mechanisms underlying their aggressive behavior may lead to better management, appropriate therapies, and good outcomes through the identification of novel specific glioma-associated genes. Members of the tripartite motif (TRIM) proteins family are involved in many biological processes, including transcriptional regulation, cell proliferation and differentiation and cell cycle progression. Alterations of TRIM proteins are associated with a variety of pathologies like developmental disorders, inflammatory diseases and cancers. Among TRIMs protein family, TRIM8 encodes an E3 ubiquitin ligase involved in various pathological processes, including hypertrophy, antiviral defense, encephalopathy, and cancer development. We have identified TRIM8 as a gene aberrantly expressed in gliomas, whose expression correlates with unfavorable clinical outcome in glioma patients. To gain insights into the TRIM8 functions, we profiled the TRIM8 transcriptome and interactome in primary mouse embryonic neural stem cells using RNA-sequencing and proteomics, followed by bioinformatics analysis. Functional analysis, including biochemical and cellular assays were then performed to explore TRIM8 roles in different pathways. Our study firstly identified enriched pathways related to the neurotransmission and to the central nervous system (CNS) functions, providing additional evidence about the existence of a functional interactive crosstalk between TRIM8 and STAT3 with possible implications in the development and progression of glioma. Then, we found that TRIM8 interacts with KIFC1 and KIF11/Eg5, two master regulators of mitotic spindle assembly and cytoskeleton reorganization. Exploring the TRIM8 role in the mitotic spindle machinery, we showed that TRIM8 localizes at the mitotic spindle during mitosis and plays a role in centrosome separation at the beginning of mitosis with a subsequent delay of the mitotic progression and impact on chromosomal stability. Our results substantiate the role of TRIM8 in the brain functions through the deregulation of genes involved in different CNS-related pathways, including JAK-STAT. Moreover, we provided insights on the physiological function of TRIM8 in the mitotic spindle machinery, pointing to an emerging role for TRIM8 in the regulation of mitosi

Advancements in Focal Amplification Detection in Tumor/Liquid Biopsies and Emerging Clinical Applications

Focal amplifications (FAs) are crucial in cancer research due to their significant diagnostic, prognostic, and therapeutic implications. FAs manifest in various forms, such as episomes, double minute chromosomes, and homogeneously staining regions, arising through different mechanisms and mainly contributing to cancer cell heterogeneity, the leading cause of drug resistance in therapy. Numerous wet-lab, mainly FISH, PCR-based assays, next-generation sequencing, and bioinformatics approaches have been set up to detect FAs, unravel the internal structure of amplicons, assess their chromatin compaction status, and investigate the transcriptional landscape associated with their occurrence in cancer cells. Most of them are tailored for tumor samples, even at the single-cell level. Conversely, very limited approaches have been set up to detect FAs in liquid biopsies. This evidence suggests the need to improve these non-invasive investigations for early tumor detection, monitoring disease progression, and evaluating treatment response. Despite the potential therapeutic implications of FAs, such as, for example, the use of HER2-specific compounds for patients with ERBB2 amplification, challenges remain, including developing selective and effective FA-targeting agents and understanding the molecular mechanisms underlying FA maintenance and replication. This review details a state-of-the-art of FA investigation, with a particular focus on liquid biopsies and single-cell approaches in tumor samples, emphasizing their potential to revolutionize the future diagnosis, prognosis, and treatment of cancer patients

IGFBP-6: At the Crossroads of Immunity, Tissue Repair and Fibrosis

Insulin-like growth factors binding protein-6 (IGFBP-6) is involved in a relevant number of cellular activities and represents an important factor in the immune response, particularly in human dendritic cells (DCs). Over the past several years, significant insights into the IGF-independent effects of IGFBP-6 were discovered, such as the induction of chemotaxis, capacity to increase oxidative burst and neutrophils degranulation, ability to induce metabolic changes in DCs, and, more recently, the regulation of the Sonic Hedgehog (SHH) signaling pathway during fibrosis. IGFBP-6 has been implicated in different human diseases, and it plays a rather controversial role in the biology of tumors. Notably, well established relationships between immunity, stroma activity, and fibrosis are prognostic and predictive of response to cancer immunotherapy. This review aims at describing the current understanding of mechanisms that link IGFBP-6 and fibrosis development and at highlighting the multiple roles of IGFBP-6 to provide an insight into evolutionarily conserved mechanisms that can be relevant for inflammation, tumor immunity, and immunological diseases

Genetic Perturbation of Pyruvate Dehydrogenase Kinase 1 Modulates Growth, Angiogenesis and Metabolic Pathways in Ovarian Cancer Xenografts

Pyruvate dehydrogenase kinase 1 (PDK1) blockade triggers are well characterized in vitro metabolic alterations in cancer cells, including reduced glycolysis and increased glucose oxidation. Here, by gene expression profiling and digital pathology-mediated quantification of in situ markers in tumors, we investigated effects of PDK1 silencing on growth, angiogenesis and metabolic features of tumor xenografts formed by highly glycolytic OC316 and OVCAR3 ovarian cancer cells. Notably, at variance with the moderate antiproliferative effects observed in vitro, we found a dramatic negative impact of PDK1 silencing on tumor growth. These findings were associated with reduced angiogenesis and increased necrosis in the OC316 and OVCAR3 tumor models, respectively. Analysis of viable tumor areas uncovered increased proliferation as well as increased apoptosis in PDK1-silenced OVCAR3 tumors. Moreover, RNA profiling disclosed increased glucose catabolic pathways—comprising both oxidative phosphorylation and glycolysis—in PDK1-silenced OVCAR3 tumors, in line with the high mitotic activity detected in the viable rim of these tumors. Altogether, our findings add new evidence in support of a link between tumor metabolism and angiogenesis and remark on the importance of investigating net effects of modulations of metabolic pathways in the context of the tumor microenvironment

Genetic Perturbation of Pyruvate Dehydrogenase Kinase 1 Modulates Growth, Angiogenesis and Metabolic Pathways in Ovarian Cancer Xenografts

Pyruvate dehydrogenase kinase 1 (PDK1) blockade triggers are well characterized in vitro metabolic alterations in cancer cells, including reduced glycolysis and increased glucose oxidation. Here, by gene expression profiling and digital pathology-mediated quantification of in situ markers in tumors, we investigated effects of PDK1 silencing on growth, angiogenesis and metabolic features of tumor xenografts formed by highly glycolytic OC316 and OVCAR3 ovarian cancer cells. Notably, at variance with the moderate antiproliferative effects observed in vitro, we found a dramatic negative impact of PDK1 silencing on tumor growth. These findings were associated with reduced angiogenesis and increased necrosis in the OC316 and OVCAR3 tumor models, respectively. Analysis of viable tumor areas uncovered increased proliferation as well as increased apoptosis in PDK1-silenced OVCAR3 tumors. Moreover, RNA profiling disclosed increased glucose catabolic pathways—comprising both oxidative phosphorylation and glycolysis—in PDK1-silenced OVCAR3 tumors, in line with the high mitotic activity detected in the viable rim of these tumors. Altogether, our findings add new evidence in support of a link between tumor metabolism and angiogenesis and remark on the importance of investigating net effects of modulations of metabolic pathways in the context of the tumor microenvironment

TRIM8-driven transcriptomic profile of neural stem cells identified glioma-related nodal genes and pathways

Background We recently reported TRIM8, encoding an E3 ubiquitin ligase, as a gene aberrantly expressed in glioblastoma whose expression suppresses cell growth and induces a significant reduction of clonogenic potential in glioblastoma cell lines. Methods we provided novel insights on TRIM8 functions by profiling the transcriptome of TRIM8-expressing primary mouse embryonal neural stem cells by RNA-sequencing and bioinformatic analysis. Functional analysis including luciferase assay, western blot, PCR arrays, Real time quantitative PCR were performed to validate the transcriptomic data. Results Our study identified enriched pathways related to the neurotransmission and to the central nervous system (CNS) functions, including axonal guidance, GABA receptor, Ephrin B, synaptic long-term potentiation/depression, and glutamate receptor signalling pathways. Finally, we provided additional evidence about the existence of a functional interactive crosstalk between TRIM8 and STAT3. Conclusions Our results substantiate the role of TRIM8 in the brain functions through the dysregulation of genes involved in different CNS-related pathways, including JAK-STAT. General significance This study provides novel insights on the physiological TRIM8 function by profiling for the first time the primary Neural Stem Cell over-expressing TRIM8 by using RNA-Sequencing methodology