Discovery of a small molecule that inhibits Bcl-3-mediated cyclin D1 expression in melanoma cells.

Molecular targeted therapy using a drug that suppresses the growth and spread of cancer cells via inhibition of a specific protein is a foundation of precision medicine and treatment. High expression of the proto-oncogene Bcl-3 promotes the proliferation and metastasis of cancer cells originating from tissues such as the colon, prostate, breast, and skin. The development of novel drugs targeting Bcl-3 alone or in combination with other therapies can cure these patients or prolong their survival. As a proof of concept, in the present study, we focused on metastatic melanoma as a model system. High-throughput screening and in vitro experiments identified BCL3ANT as a lead molecule that could interfere with Bcl-3-mediated cyclin D1 expression and cell proliferation and migration in melanoma. In experimental animal models of melanoma, it was demonstrated that the use of a Bcl-3 inhibitor can influence the survival of melanoma cells. Since there are no other inhibitors against Bcl-3 in the clinical pipeline for cancer treatment, this presents a unique opportunity to develop a highly specific drug against malignant melanoma to meet an urgent clinical need.

From master's thesis to research publication: a mixed-methods study of medical student publishing and experiences with the publishing process.

BACKGROUND: Medical student master's theses are often carried out as research projects, and some are published as research papers in journals. We investigated the percentage of master's theses conducted by 5th -year students at the Medical Degree Program at Lund University, Sweden, that subsequently served as the basis for research publications. In addition, we explored both student and supervisor experiences with the publishing process. METHODS: A cohort of four semesters of student data covering the period from 2019 to 2020 (n = 446) was searched in PubMed, Embase and the Web of Science to assess whether they had been published as research papers. Surveys were sent to students (n = 121) and supervisors (n = 77) to explore their experiences with the publishing process. RESULTS: We found that 33% (149 of 446) of the students in the 2019-2020 cohort subsequently published their theses, and 50% of these students were listed as first authors. Most students published original research. Students (n = 21) and supervisors (n = 44) reported that the publishing process was time-consuming and that students needed multilevel support from supervisors to achieve successful publication. The publishing process was reported by 79% of the students to have led to additional learning. Most of the papers (126 of 149, 85%) had a clinical or patient-oriented focus. CONCLUSION: A high percentage of the student publications in which students are listed as first authors require engagement from both students and supervisors. Supervisors play an essential role in supporting students in a successful publication process. Most of the published papers were either clinical or patient-oriented research.

The E3 ligase Itch and deubiquitinase Cyld act together to regulate Tak1 and inflammation.

Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. Here we demonstrate that E3 ligase Itch and deubiquitinase Cyld formed a complex via interaction through 'WW-PPXY' motifs. The Itch-Cyld complex sequentially cleaved Lys63-linked ubiquitin chains and catalyzed Lys48-linked ubiquitination on the kinase Tak1 to terminate inflammatory signaling via tumor necrosis factor. Reconstitution of wild-type Cyld but not the mutant Cyld(Y485A), which cannot associate with Itch, blocked sustained Tak1 activation and proinflammatory cytokine production by Cyld(-/-) bone marrow-derived macrophages. Deficiency in Itch or Cyld led to chronic production of tumor-promoting cytokines by tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have identified an Itch-Cyld-mediated regulatory mechanism in innate inflammatory cells.

Tetraploidization Increases the Motility and Invasiveness of Cancer Cells.

Polyploidy and metastasis are associated with a low probability of disease-free survival in cancer patients. Polyploid cells are known to facilitate tumorigenesis. However, few data associate polyploidization with metastasis. Here, by generating and using diploid (2n) and tetraploid (4n) clones from malignant fibrous histiocytoma (MFH) and colon carcinoma (RKO), we demonstrate the migration and invasion advantage of tetraploid cells in vitro using several assays, including the wound healing, the OrisTM two-dimensional cell migration, single-cell migration tracking by video microscopy, the Boyden chamber, and the xCELLigence RTCA real-time cell migration. Motility advantage was observed despite tetraploid cell proliferation weakness. We could also demonstrate preferential metastatic potential in vivo for the tetraploid clone using the tail vein injection in mice and tracking metastatic tumors in the lung. Using the Mitelman Database of Chromosome Aberrations in Cancer, we found an accumulation of polyploid karyotypes in metastatic tumors compared to primary ones. This work reveals the clinical relevance of the polyploid subpopulation and the strategic need to highlight polyploidy in preclinical studies as a therapeutic target for metastasis.

The roles of interleukin-1 receptor accessory protein in certain inflammatory conditions.

Interleukin-1 receptor accessory protein (IL-1RAcP) is a member of the immunoglobulin superfamily proteins consisting of soluble and membranous isoforms. IL-1RAcP plays an essential role in the signaling of the IL-1 family cytokines such as IL-1, IL-33 and IL-36, as well as tyrosine kinases FLT3 and C-Kit. IL-1RAcP generally initiates inflammatory signaling pathway through the recruitment of signaling mediators, including MYD88 and IRAK. Chronic inflammation following prolonged signaling of cytokine receptors is a critical process in the pathogenesis of many inflammatory disorders, including autoimmunity, obesity, psoriasis, type 1 diabetes, endometriosis, preeclampsia and Alzheimer's disease. Recently IL-1RAcP aberrant signaling has been considered to play a central role in the pathogenesis of these chronic inflammatory diseases. Targeting IL-1RAcP signaling pathway that was recently considered in clinical trials related to malignancies also indicates its potential as therapeutic target for the inflammatory and autoimmune diseases. This review summarizes the molecular structure, components associated with IL-1RAcP signaling pathways, and their involvement in the pathogenesis of different inflammatory diseases. We will also discuss the effect of IL-1RAcP inhibition for treatment proposes.

Deletion of Nemo-like Kinase in T Cells Reduces Single-Positive CD8+ Thymocyte Population.

The ß-catenin/Wnt signaling pathway plays an important role in all stages of T cell development. Nemo-like kinase (NLK) is an evolutionary conserved serine/threonine kinase and a negative regulator of the Wnt signaling pathway. NLK can directly phosphorylate histone deacetylase 1 (HDAC1), as well as T cell factor/lymphoid enhancer-binding factor (TCF/LEF), causing subsequent repression of target gene transcription. By engineering mice lacking NLK in early stages of T cell development, we set out to characterize the role NLK plays in T cell development and found that deletion of NLK does not affect mouse health or lymphoid tissue development. Instead, these mice harbored a reduced number of single-positive (SP) CD8+ thymocytes without any defects in the SP CD4+ thymocyte population. The decrease in SP CD8+ thymocytes was not caused by a block in differentiation from double-positive CD4+CD8+ cells. Neither TCR signaling nor activation was altered in the absence of NLK. Instead, we observed a significant increase in cell death and reduced phosphorylation of LEF1 as well as HDAC1 among NLK-deleted SP CD8+ cells. Thus, NLK seems to play an important role in the survival of CD8+ thymocytes. Our data provide evidence for a new function for NLK with regard to its involvement in T cell development and supporting survival of SP CD8+ thymocytes.

Preferential Killing of Tetraploid Colon Cancer Cells by Targeting the Mitotic Kinase PLK1.

BACKGROUND/AIMS: Chromosomal instability is a well-known factor in the progression of different types of cancer, including colorectal cancer. Chromosomal instability results in severely rearranged karyotypes and aneuploidy. Tetraploidy constitutes an intermediate phase during the polyploidy/aneuploidy cascade in oncogenesis, and tetraploid cells are particularly resistant to chemotherapy. Whether inhibition of the mitotic protein polo-like kinase 1 (PLK1) prevents the survival of tetraploid colon cancer cells is unknown. METHODS: Diploid and tetraploid cells were transfected with siPLK1 or treated with PLK1 inhibitor Bi2536 in combination with spindle poison. Cell toxicity was assessed via crystal violet staining and clonogenic assay. Flow cytometry assessment analyzed numerous cell apoptotic parameters and cell cycle phases. Synergistic activity between Bi2536 and paclitaxel, vincristine or colchicine was calculated using the CompuSyn software. RESULTS: Inhibition or abrogation of PLK1 prevented the survival of colon cancer cells, specifically tetraploid cells. The cell death induced by PLK inhibition was due to mitotic slippage, followed by the activation of the intrinsic pathway of apoptosis. We further demonstrated that co-treatment of the tetraploid colon cancer cells with a PLK1 inhibitor and the microtubule polymerisation inhibitor vincristine or colchicine, but not the microtubule depolymerisation inhibitor paclitaxel, provoked a lethal synergistic effect. CONCLUSION: PLK1 inhibition together with microtubule-targeting chemicals, serve as a potent therapeutic strategy for targeting tetraploid cancer cells.

Nemo-Like Kinase in Development and Diseases: Insights from Mouse Studies.

The Wnt signalling pathway is a central communication cascade between cells to orchestrate polarity and fate during development and adult tissue homeostasis in various organisms. This pathway can be regulated by different signalling molecules in several steps. One of the coordinators in this pathway is Nemo-like kinase (NLK), which is an atypical proline-directed serine/threonine mitogen-activated protein (MAP) kinase. Very recently, NLK was established as an essential regulator in different cellular processes and abnormal NLK expression was highlighted to affect the development and progression of various diseases. In this review, we focused on the recent discoveries by using NLK-deficient mice, which show a phenotype in the development and function of organs such as the lung, heart and skeleton. Furthermore, NLK could conduct the function and differentiation of cells from the immune system, in addition to regulating neurodegenerative diseases, such as Huntington's disease and spinocerebellar ataxias. Overall, generations of NLK-deficient mice have taught us valuable lessons about the role of this kinase in certain diseases and development.

Gene Expression Signature of Acquired Chemoresistance in Neuroblastoma Cells.

Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. Patients with relapsed disease have a poor prognosis despite intense treatment. In the present study, we aimed to identify chemoresistance gene expression signatures in vincristine resistant neuroblastoma cells. We found that vincristine-resistant neuroblastoma cells formed larger clones and survived under reduced serum conditions as compared with non-resistant parental cells. To identify the possible mechanisms underlying vincristine resistance in neuroblastoma cells, we investigated the expression profiles of genes known to be involved in cancer drug resistance. This specific gene expression patterns could predict the behavior of a tumor in response to chemotherapy and for predicting the prognosis of high-risk neuroblastoma patients. Our signature could help chemoresistant neuroblastoma patients in avoiding useless and harmful chemotherapy cycles.

Discovery of epi-Enprioline as a Novel Drug for the Treatment of Vincristine Resistant Neuroblastoma.

Neuroblastoma is a childhood solid tumour originating from undifferentiated neural progenitor cells of the sympathetic nervous system. Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. In the present study, we aimed to identify novel drugs that can inhibit the growth and survival of chemoresistant neuroblastoma. High-throughput screening identified a small molecule, epi-enprioline that was able to induce apoptosis of vincristine-resistant neuroblastoma cells via the mitochondrial apoptotic pathway. Epi-enprioline reduced tumour growth in multiple preclinical models, including an orthotopic neuroblastoma patient-derived xenograft model in vivo. In summary, our data suggest that epi-enprioline can be considered as a lead compound for the treatment of vincristine-resistant neuroblastoma uncovering a novel strategy, which can be further explored as a treatment for drug-resistant neuroblastoma.

Cylindromatosis--A Protective Molecule against Liver Diseases.

Cylindromatosis (CYLD) is a deubiquitination enzyme involved in the regulation of different cellular processes including inflammation, fibrosis, and cancer. The function of CYLD is via deubiquitination of specific substrates in different signaling pathways including NF-κB, Notch, and JNK. CYLD contributes to hepatic homeostasis and restoration upon liver injury. Mutation or disruption of the activity of CYLD in animals aggravates acute as well as chronic liver injury and promotes development and progression of hepatocellular cancer. This is mediated by a shift of the balance toward pro-inflammatory, pro-fibrogenic, and pro-oncogenic pathways. In this review, we will explain the liver-associated signaling pathways that CYLD regulates in hepatocytes and nonparenchymal liver cells under physiological and pathological conditions. We will also describe the most recent findings concerning CYLD-mediated downstream signaling in the liver in situations such as injury, infection, inflammation, and cancer. Furthermore, we will discuss the potential of novel diagnostic tools and treatment strategies utilizing CYLD and its target genes.

Putative role of SUMOylation in controlling the activity of deubiquitinating enzymes in cancer.

Deubiquitinating enzymes (DUBs) are specialized proteins that can recognize ubiquitinated proteins, and after direct interaction, deconjugate monomeric or polymeric ubiquitin chains, thus changing the fate of the substrates. This process is instrumental in mediating or changing downstream signaling pathways. Beside mutations and alterations in their expression levels, the activity and stability of deubiquitinating enzymes is vital for their function. SUMOylations consist of the conjugation of the small peptide SUMO to protein substrates which is very similar to ubiquitination in the mechanistic and machinery required. In this review, we will focus on how SUMOylation can regulate DUB enzymatic activity, stability or DUB interaction with partners and substrates, in cancer. Furthermore, we will discuss the impact of these recent findings in the identification of new potential tools for efficient anticancer treatment strategies.

Multifaceted role of the ubiquitin ligase Itch in immune regulation.

The HECT-type E3 ligase Itch is increasingly being shown to have a vital role in immune regulation. Itch deficiency leads to deleterious inflammatory disorders both in mice and humans. By adding ubiquitin to the key signaling intermediates, Itch functions as a critical regulator of lymphocyte-cell activation, differentiation and immune tolerance. Also, Itch cooperates with deubiquitinating enzymes such as A20 and Cyld to terminate NF-κB signaling and prevent chronic inflammation. This review summarizes recent advances that highlight Itch's role in lymphocyte function and explores recent insights regarding its role as a regulator of inflammatory signaling.

CYLD enhances severe listeriosis by impairing IL-6/STAT3-dependent fibrin production.

The facultative intracellular bacterium Listeria monocytogenes (Lm) may cause severe infection in humans and livestock. Control of acute listeriosis is primarily dependent on innate immune responses, which are strongly regulated by NF-κB, and tissue protective factors including fibrin. However, molecular pathways connecting NF-κB and fibrin production are poorly described. Here, we investigated whether the deubiquitinating enzyme CYLD, which is an inhibitor of NF-κB-dependent immune responses, regulated these protective host responses in murine listeriosis. Upon high dose systemic infection, all C57BL/6 Cyld(-/-) mice survived, whereas 100% of wildtype mice succumbed due to severe liver pathology with impaired pathogen control and hemorrhage within 6 days. Upon in vitro infection with Lm, CYLD reduced NF-κB-dependent production of reactive oxygen species, interleukin (IL)-6 secretion, and control of bacteria in macrophages. Furthermore, Western blot analyses showed that CYLD impaired STAT3-dependent fibrin production in cultivated hepatocytes. Immunoprecipitation experiments revealed that CYLD interacted with STAT3 in the cytoplasm and strongly reduced K63-ubiquitination of STAT3 in IL-6 stimulated hepatocytes. In addition, CYLD diminished IL-6-induced STAT3 activity by reducing nuclear accumulation of phosphorylated STAT3. In vivo, CYLD also reduced hepatic STAT3 K63-ubiquitination and activation, NF-κB activation, IL-6 and NOX2 mRNA production as well as fibrin production in murine listeriosis. In vivo neutralization of IL-6 by anti-IL-6 antibody, STAT3 by siRNA, and fibrin by warfarin treatment, respectively, demonstrated that IL-6-induced, STAT3-mediated fibrin production significantly contributed to protection in Cyld(-/-) mice. In addition, in vivo Cyld siRNA treatment increased STAT3 phosphorylation, fibrin production, pathogen control and survival of Lm-infected WT mice illustrating that therapeutic inhibition of CYLD augments the protective NF-κB/IL-6/STAT3 pathway and fibrin production.

Cylindromatosis gene CYLD regulates hepatocyte growth factor expression in hepatic stellate cells through interaction with histone deacetylase 7.

UNLABELLED: Hepatic fibrosis is considered as a physiological wound-healing response to liver injury. The process involves several factors, such as hepatocyte growth factor (HGF), which restrains hepatic injury and facilitates reversibility of fibrotic reaction in response to an acute insult. Chronic liver injury and sustained inflammation cause progressive fibrosis and, ultimately, organ dysfunction. The mechanisms tipping the balance from restoration to progressive liver tissue scarring are not well understood. In the present study, we identify a mechanism in which the tumor-suppressor gene, cylindromatosis (CYLD), confers protection from hepatocellular injury and fibrosis. Mice lacking CYLD (CYLD-/-) were highly susceptible to hepatocellular damage, inflammation, and fibrosis and revealed significantly lower hepatic HGF levels, compared to wild-type (WT) animals. Exogenous application of HGF rescued the liver injury phenotype of CYLD-/- mice. In the absence of CYLD, gene transcription of HGF in hepatic stellate cells was repressed through binding of histone deacetylase 7 (HDAC7) to the promoter of HGF. In WT cells, CYLD removed HDAC7 from the HGF promoter and induced HGF expression. Of note, this interaction occurred independently of the deubiquitinating activity of CYLD. CONCLUSIONS: Our findings highlight a novel link between CYLD and HDAC7, offering mechanistic insight into the contribution of these proteins to progression of liver disease. Thus, through regulation of HGF level, CYLD ameliorates hepatocellular damage and liver fibrogenesis.

Early diagnostic value of Bcl-3 localization in colorectal cancer.

BACKGROUND: B-cell leukemia 3 (Bcl-3) is a member of the inhibitor of κB family, which regulates a wide range of biological processes by functioning as a transcriptional activator or as a repressor of target genes. Elevated expression, sustained nuclear accumulation, and uncontrolled activation of Bcl-3 causes increased cellular proliferation or survival, dependent on the tissue and type of stimuli. METHODS: We retrospectively reviewed patients who were diagnosed with colorectal cancer at Skåne University Hospital in Malmö between 1st of January 1990 and 31st of December 1991. Bcl-3 localization in colorectal cancer was assessed by immunohistochemistry on tissue microarray and freshly isolated colon from patients. Correlation between Bcl-3 localization and clinicopathological parameters of the cohort were evaluated using the Spearman rank-order correlation coefficient. In addition, Bcl-3 expression and localization in colon adenocarcinoma cells were analysed by western blot, immunohistochemistry and subcellular fractionation separately. RESULTS: We found that Bcl-3 was mainly localized in the cytoplasm in the tumour tissue isolated from colon cancer patients. Normal colon samples from the same patients showed Bcl-3 localization in the nucleus. In three out of six colon cancer cell lines, we detected elevated levels of Bcl-3. In these cell lines Bcl-3 was accumulated in the cytosol. We confirmed these findings by analysing Bcl-3 localization in a colon tissue micro array consisting of 270 cases. In these samples Bcl-3 localization correlated with the proliferation marker Ki-67, but not with the apoptotic marker Caspase 3. CONCLUSION: These findings indicate that analysis of the subcellular localization of Bcl-3 could be a potential-early diagnostic marker in colon cancer.

Ubiquitin chain cleavage: CYLD at work.

The tumor suppressor CYLD is a deubiquitylating enzyme that negatively regulates different signaling pathways by removing lysine 63-linked polyubiquitin chains from several specific substrates. In various tumor types, CYLD loss can lead to cell survival or cell proliferation. In addition to its loss due to mutations, CYLD expression can also be decreased through transcriptional and post-transcriptional regulatory mechanisms. Moreover, as epigenetic repression of CYLD can affect tumor progression in different cancer types, the activation of the CYLD promoter ensures the tight control of an inflammatory response. Recent work also shows that CYLD activity can be governed by different regulatory mechanisms including phosphorylation, thus providing another layer of control for diverse physiological processes.

CYLD controls c-MYC expression through the JNK-dependent signaling pathway in hepatocellular carcinoma.

Posttranslational modification of different proteins via direct ubiquitin attachment is vital for mediating various cellular processes. Cylindromatosis (CYLD), a deubiquitination enzyme, is able to cleave the polyubiquitin chains from the substrate and to regulate different signaling pathways. Loss, or reduced expression, of CYLD is observed in different types of human cancer, such as hepatocellular carcinoma (HCC). However, the molecular mechanism by which CYLD affects cancerogenesis has to date not been unveiled. The aim of the present study was to examine how CYLD regulates cellular functions and signaling pathways during hepatocancerogenesis. We found that mice lacking CYLD were highly susceptible to chemically induced liver cancer. The mechanism behind proved to be an elevated proliferation rate of hepatocytes, owing to sustained c-Jun N-terminal kinase 1 (JNK1)-mediated signaling via ubiquitination of TNF receptor-associated factor 2 and expression of c-MYC. Overexpression of wild-type CYLD in HCC cell lines prevented cell proliferation, without affecting apoptosis, adhesion and migration. A combined immunohistochemical and tissue microarray analysis of 81 human HCC tissues revealed that CYLD expression is negatively correlated with expression of proliferation markers Ki-67 and c-MYC. To conclude, we found that downregulation of CYLD induces tumor cell proliferation, consequently contributing to the aggressive growth of HCC. Our findings suggest that CYLD holds potential to serve as a marker for HCC progression, and its link to c-MYC via JNK1 may provide the foundation for new therapeutic strategies for HCC patients.

CYLD negatively regulates cell-cycle progression by inactivating HDAC6 and increasing the levels of acetylated tubulin.

CYLD is a tumour-suppressor gene that is mutated in a benign skin tumour syndrome called cylindromatosis. The CYLD gene product is a deubiquitinating enzyme that was shown to regulate cell proliferation, cell survival and inflammatory responses, mainly through inhibiting NF-kappaB signalling. Here we show that CYLD controls cell growth and division at the G(1)/S-phase as well as cytokinesis by associating with alpha-tubulin and microtubules through its CAP-Gly domains. Translocation of activated CYLD to the perinuclear region of the cell is achieved by an inhibitory interaction of CYLD with histone deacetylase-6 (HDAC6) leading to an increase in the levels of acetylated alpha-tubulin around the nucleus. This facilitates the interaction of CYLD with Bcl-3, leading to a significant delay in the G(1)-to-S-phase transition. Finally, CYLD also interacts with HDAC6 in the midbody where it regulates the rate of cytokinesis in a deubiquitinase-independent manner. Altogether these results identify a mechanism by which CYLD regulates cell proliferation at distinct cell-cycle phases.