OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination

Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de-ubiquitinase OTUB1 as a negative regulator of RAS mono- and di-ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild-type RAS cells. An increase of OTUB1 expression is commonly observed in non-small-cell lung carcinomas harboring wild-type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer development

PRAME Expression in Mucosal Melanoma of the Head and Neck Region

PRAME (PReferentially expressed Antigen in MElanoma), a cancer-testis antigen expressed in normal and neoplastic tissues with several functions, proved to be a useful diagnostic tool in the differential diagnosis between benign and malignant melanocytic lesions. The current study aims to perform PRAME stain on a retrospective case series of mucosal melanocytic tumors of the head and neck region to compare 3 different scores and evaluate the most reliable one in this diagnostic set. Immunohistochemical analysis for PRAME was performed in 54 benign and malignant mucosal melanocytic tumors of the head and neck region collected from 41 patients. The best-performing cutoff of PRAME-positive cells (nuclear stain) to differentiate benign and malignant mucosal melanocytic tumors of the head and neck region is that proposed by Raghavan and colleagues (<60%/≥60% of PRAME-positive cells), with 100% and 77.8% of benign lesions and malignant tumors respectively correctly identified. Applying this score, PRAME stain showed the best results (sensitivity, specificity, accuracy, and positive and negative predictive values) for the diagnosis of head and neck melanocytic tumors. However, a subset of PRAME-negative malignant tumors was identified, especially located in the palatal area (hard and soft palate). Finally, high PRAME expression (≥60%) was associated with specific sites (nasal cavity/nasal septum/turbinates nasopharynx, and the maxillary sinus), nodular histotype, and female sex

The mitotic checkpoint is a targetable vulnerability of carboplatin-resistant triple negative breast cancers

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, lacking effective therapy. Many TNBCs show remarkable response to carboplatin-based chemotherapy, but often develop resistance over time. With increasing use of carboplatin in the clinic, there is a pressing need to identify vulnerabilities of carboplatin-resistant tumors. In this study, we generated carboplatin-resistant TNBC MDA-MB-468 cell line and patient derived TNBC xenograft models. Mass spectrometry-based proteome profiling demonstrated that carboplatin resistance in TNBC is linked to drastic metabolism rewiring and upregulation of anti-oxidative response that supports cell replication by maintaining low levels of DNA damage in the presence of carboplatin. Carboplatin-resistant cells also exhibited dysregulation of the mitotic checkpoint. A kinome shRNA screen revealed that carboplatin-resistant cells are vulnerable to the depletion of the mitotic checkpoint regulators, whereas the checkpoint kinases CHEK1 and WEE1 are indispensable for the survival of carboplatin-resistant cells in the presence of carboplatin. We confirmed that pharmacological inhibition of CHEK1 by prexasertib in the presence of carboplatin is well tolerated by mice and suppresses the growth of carboplatin-resistant TNBC xenografts. Thus, abrogation of the mitotic checkpoint by CHEK1 inhibition re-sensitizes carboplatin-resistant TNBCs to carboplatin and represents a potential strategy for the treatment of carboplatin-resistant TNBCs

OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination

Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de-ubiquitinase OTUB1 as a negative regulator of RAS mono- and di-ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild-type RAS cells. An increase of OTUB1 expression is commonly observed in non-small-cell lung carcinomas harboring wild-type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer developmen

Stress-induced lncRNA LASTR fosters cancer cell fitness by regulating the activity of the U4/U6 recycling factor SART3

Dysregulated splicing is a common event in cancer even in the absence of mutations in the core splicing machinery. The aberrant long non-coding transcriptome constitutes an uncharacterized level of regulation of post-transcriptional events in cancer. Here, we found that the stress-induced long non-coding RNA (lncRNA), LINCO2657 or LASTR (lncRNA associated with SART3 regulation of splicing), is upregulated in hypoxic breast cancer and is essential for the growth of LASTR-positive triple-negative breast tumors. LASTR is upregulated in several types of epithelial cancers due to the activation of the stress-induced JNK/c-JUN pathway. Using a mass-spectrometry based approach, we identified the RNA-splicing factor SART3 as a LASTR-interacting partner. We found that LASTR promotes splicing efficiency by controlling SART3 association with the U4 and U6 small nuclear ribonucleoproteins (snRNP) during spliceosome recycling. Intron retention induced by LASTR depletion downregulates expression of essential genes, ultimately decreasing the fitness of cancer cells

Syndecan-syntenin regulates the biogenesis of exosomes

Syndecans, heparan sulfate proteoglycans, are essential for fine-regulating signalling events between cells. The extracellular heparan sulfate chains of these membrane proteins attract several signalling molecules, such as morphogens, to cell surfaces, but the mechanisms of their regulatory functions remain largely unknown. We have found that, through the PDZ protein syntenin, a well-known syndecan cytoplasmic adaptor, syndecans are connected to ALIX, an auxiliary component of the ESCRT machinery, implicated in vesicular budding, and marker of small signalling vesicles released by cells known as exosomes . Here, we explore whether the syndecan-syntenin-ALIX connection might regulate the biogenesis of exosomes, impacting on the cellular release of morphogen signals.Syndecan 1 is released in exosomes and co-fractionates, together with syntenin and ALIX, with exosomal marker proteins such as CD63. While syntenin over-expression results, depending on ALIX, in an increased release of exosomes, the depletion of syntenin (or ALIX) by RNAi treatment blocks the exosomal accumulation of syndecan and marker proteins. Strikingly, syndecan RNAi markedly suppresses exosome production. Syntenin has an effect on a subset of exosomes: while CD63 and syntenin largely reside in the same exosome population, as shown by immuno-fractionation and by fluorescence cross-correlation spectroscopy, flotillin-1 co-purifies mainly with CD63-negative vesicles and its release is not affected by syntenin. Syntenin-exosomes are of endosomal origin and depend on endosomal trafficking, as shown by RNAi targeting RAB7, a small GTPase regulating late endocytic membrane traffic. Electron microscopy analysis of syntenin-depleted cells shows that MVBs are largely devoid of intraluminal vesicles, indicating that syntenin supports the intraluminal budding and then production of exosomes. The molecular machinery responsible for the biogenesis of syntenin-exosomes is ESCRT-dependent, as depletion by RNAi of several components of the ESCRT complexes affects the formation of these exosomes. The presence of heparan sulfate chains on syndecans is necessary for producing exosomes, and the release of exosomes, impaired in HS-depleted cells, can be rescued by anti-syndecan antibody, suggesting that ligand-initiated syndecan oligomerization might be the driving force for the production of syntenin-exosomes. Syndecan-syntenin exosomes contain HS-dependent signalling cargo, such as p-FGFR, and can transfer FGF-responsiveness to recipient cells, hinting at the functional relevance of these exosomes.The results obtained show that interfering with syndecan-syntenin-ALIX reduces exosome production by affecting the biogenesis of MVBs and that syndecan-syntenin-ALIX complexes control the composition of a specific population of exosomes, potentially transferring specific information between cells. The molecular connection of syndecan with exosomes might thus support a novel role for proteoglycans in vesicular trafficking and trans-cellular signalling, supporting the release of growth factors and signalling components into the extracellular space. The establishment of a direct link between syndecans and exosomes, as a novel mechanism of morphogen spreading, may help explain the role of HSPG in the diffusion of morphogenetic signals, in particular how lipid-modified morphogens travel long-range. Syndecan-syntenin exosomes might also participate in the biology of HS-associated pathological processes, including neurodegenerative and oncogenic diseases.status: publishe

Novel Therapeutic Approaches Targeting Post-Translational Modifications in Lung Cancer

Lung cancer is one of the most common cancers worldwide. It consists of two different subtypes: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Despite novel therapeutic options such as immunotherapy, only 20% of lung cancer patients survive the disease after five years. This low survival rate is due to acquired drug resistance and severe off-target effects caused by currently used therapies. Identification and development of novel and targeted therapeutic approaches are urgently required to improve the standard of care for lung cancer patients. Here, we describe the recent development of novel drug-delivery approaches, such as adenovirus, lipid nanoparticles, and PROTACs, that have been tested in clinical trials and experimentally in the context of fundamental research. These different options show that it is now possible to target protein kinases, phosphatases, ubiquitin ligases, or protein modifications directly in lung cancer to block disease progression. Furthermore, the recent acceptance of RNA vaccines using lipid nanoparticles has further revealed therapeutic options that could be combined with chemo-/immunotherapies to improve current lung cancer therapies. This review aims to compare recent advances in the pharmaceutical research field for the development of technologies targeting post-translational modifications or protein modifiers involved in the tumorigenesis of lung cancer

Cracking the Monoubiquitin Code of Genetic Diseases

Ubiquitination is a versatile and dynamic post-translational modification in which single ubiquitin molecules or polyubiquitin chains are attached to target proteins, giving rise to mono- or poly-ubiquitination, respectively. The majority of research in the ubiquitin field focused on degradative polyubiquitination, whereas more recent studies uncovered the role of single ubiquitin modification in important physiological processes. Monoubiquitination can modulate the stability, subcellular localization, binding properties, and activity of the target proteins. Understanding the function of monoubiquitination in normal physiology and pathology has important therapeutic implications, as alterations in the monoubiquitin pathway are found in a broad range of genetic diseases. This review highlights a link between monoubiquitin signaling and the pathogenesis of genetic disorders

The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response

The RAS-like GTPase RALB mediates cellular responses to nutrient availability or viral infection by respectively engaging two components of the exocyst complex, EXO84 and SEC5. RALB employs SEC5 to trigger innate immunity signalling, whereas RALB-EXO84 interaction induces autophagocytosis. How this differential interaction is achieved molecularly by the RAL GTPase remains unknown. We found that whereas GTP binding turns on RALB activity, ubiquitylation of RALB at Lys 47 tunes its activity towards a particular effector. Specifically, ubiquitylation at Lys 47 sterically inhibits RALB binding to EXO84, while facilitating its interaction with SEC5. Double-stranded RNA promotes RALB ubiquitylation and SEC5-TBK1 complex formation. In contrast, nutrient starvation induces RALB deubiquitylation by accumulation and relocalization of the deubiquitylase USP33 to RALB-positive vesicles. Deubiquitylated RALB promotes the assembly of the RALB-EXO84-beclin-1 complexes driving autophagosome formation. Thus, ubiquitylation within the effector-binding domain provides the switch for the dual functions of RALB in autophagy and innate immune responses.status: publishe

OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination

Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de-ubiquitinase OTUB1 as a negative regulator of RAS mono- and di-ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild-type RAS cells. An increase of OTUB1 expression is commonly observed in non-small-cell lung carcinomas harboring wild-type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer development.status: publishe