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BAP1-inactivated melanocytic tumor (BIMT) is a group of melanocytic neoplasms with epithelioid cell morphology molecularly characterized by the loss of function of BAP1, a tumor suppressor gene located on chromosome 3p21, and a mutually exclusive mitogenic driver mutation, more commonly BRAF. BIMTs can occur as a sporadic lesion or, less commonly, in the setting of an autosomal dominant cancer susceptibility syndrome caused by a BAP1 germline inactivating mutation. Owing to the frequent identification of remnants of a conventional nevus, BIMTs are currently classified within the group of combined melanocytic nevi. "Pure" lesions can also be observed. We studied 50 BIMTs from 36 patients. Most lesions were composed of epithelioid melanocytes of varying size and shapes, resulting extreme cytomorphological heterogeneity. Several distinctive morphological variants of multinucleated/giant cells were identified. Some hitherto underrecognized microscopic features, especially regarding nuclear characteristics included nuclear blebbing, nuclear budding, micronuclei, shadow nuclei, peculiar cytoplasmic projections (ant-bear cells) often containing micronuclei and cell-in-cell structures (entosis). In addition, there were mixed nests of conventional and BAP1-inactivated melanocytes and squeezed remnants of the original nevus. Of the 26 lesions studied, 24 yielded a BRAF mutation, while in the remaining two cases there was a RAF1 fusion. BAP1 biallelic and singe allele mutations were found in 4/22 and 16/24 neoplasms, respectively. In five patients, there was a BAP1 germline mutation. Six novel, previously unreported BAP1 mutations have been identified. BAP1 heterozygous loss was detected in 11/22 lesions. Fluorescence in situ hybridization for copy number changes revealed a related amplification of both RREB1 and MYC genes in one tumor, whereas the remaining 20 lesions studied were negative; no TERT-p mutation was found in 14 studied neoplasms. Tetraploidy was identified in 5/21 BIMTs. Of the 21 patients with available follow-up, only one child had a locoregional lymph node metastasis. Our results support a progression of BIMTs from a conventional BRAF mutated in which the original nevus is gradually replaced by epithelioid BAP1-inactivated melanocytes. Some features suggest more complex underlying pathophysiological events that need to be elucidated.

The HERC protein family is one of three subfamilies of Homologous to E6AP C-terminus (HECT) E3 ubiquitin ligases. Six HERC genes have been described in humans, two of which encode Large HERC proteins -HERC1 and HERC2- with molecular weights above 520 kDa that are constitutively expressed in the brain. There is a large body of evidence that mutations in these Large HERC genes produce clinical syndromes in which key neurodevelopmental events are altered, resulting in intellectual disability and other neurological disorders like epileptic seizures, dementia and/or signs of autism. In line with these consequences in humans, two mice carrying mutations in the Large HERC genes have been studied quite intensely: the tambaleante mutant for Herc1 and the Herc2+/530 mutant for Herc2. In both these mutant mice there are clear signs that autophagy is dysregulated, eliciting cerebellar Purkinje cell death and impairing motor control. The tambaleante mouse was the first of these mice to appear and is the best studied, in which the Herc1 mutation elicits: (i) delayed neural transmission in the peripheral nervous system; (ii) impaired learning, memory and motor control; and (iii) altered presynaptic membrane dynamics. In this review, we discuss the information currently available on HERC proteins in the nervous system and their biological activity, the dysregulation of which could explain certain neurodevelopmental syndromes and/or neurodegenerative diseases., This work was funded by the following grants: EMP-V (DGICYT BFU2011-27207 and Spanish Junta de Andalucía CTS-2257); RR (Spanish Junta de Andalucía BIO-113 and SAF2015-64171-R); SB (Spanish Junta de Andalucía, Postdoctoral grant PAIDI-Doctor); JAA (Spanish Junta de Andalucía BIO-209 and DGICYT BFU2015-64536-R); JLR (Agencia Estatal de Investigación: PID2020-120344RB-I00 / MCIN / AEI / 10.13039/501100011033).

Malignant pleural mesothelioma (MPM) is a rare, aggressive, and incurable cancer arising from the mesothelial lining of the pleura, with few available treatment options. We recently reported that loss of function of the nuclear deubiquitinase BRCA1-associated protein 1 (BAP1), a frequent event in MPM, is associated with sensitivity to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–mediated apoptosis. As a potential underlying mechanism, here we report that BAP1 negatively regulates the expression of TRAIL receptors: death receptor 4 (DR4) and death receptor 5 (DR5). Using tissue microarrays of tumor samples from MPM patients, we found a strong inverse correlation between BAP1 and TRAIL receptor expression. BAP1 knockdown increased DR4 and DR5 expression, whereas overexpression of BAP1 had the opposite effect. Reporter assays confirmed wt-BAP1, but not catalytically inactive BAP1 mutant, reduced promoter activities of DR4 and DR5, suggesting deubiquitinase activity is required for the regulation of gene expression. Co-immunoprecipitation studies demonstrated direct binding of BAP1 to the transcription factor Ying Yang 1 (YY1), and chromatin immunoprecipitation assays revealed BAP1 and YY1 to be enriched in the promoter regions of DR4 and DR5. Knockdown of YY1 also increased DR4 and DR5 expression and sensitivity to TRAIL. These results suggest that BAP1 and YY1 cooperatively repress transcription of TRAIL receptors. Our finding that BAP1 directly regulates the extrinsic apoptotic pathway will provide new insights into the role of BAP1 in the development of MPM and other cancers with frequent BAP1 mutations.

Hennekam lymphangiectasia-lymphedema syndrome has been linked to single-nucleotide polymorphisms in the CCBE1 (collagen and calcium-binding EGF domains 1) gene. Several bioinformatics methods were used to find the most dangerous nsSNPs that could affect CCBE1 structure and function. Using state-of-the-art in silico tools, this study examined the most pathogenic nonsynonymous single-nucleotide polymorphisms (nsSNPs) that disrupt the CCBE1 protein and extracellular matrix remodeling and migration. Our results indicate that seven nsSNPs, rs115982879, rs149792489, rs374941368, rs121908254, rs149531418, rs121908251, and rs372499913, are deleterious in the CCBE1 gene, four (G330E, C102S, C174R, and G107D) of which are the highly deleterious, two of them (G330E and G107D) have never been seen reported in the context of Hennekam syndrome. Twelve missense SNPs, rs199902030, rs267605221, rs37517418, rs80008675, rs116596858, rs116675104, rs121908252, rs147974432, rs147681552, rs192224843, rs139059968, and rs148498685, are found to revert into stop codons. Structural homology-based methods and sequence homology-based tools revealed that 8.8% of the nsSNPs are pathogenic. SIFT, PolyPhen2, M-CAP, CADD, FATHMM-MKL, DANN, PANTHER, Mutation Taster, LRT, and SNAP2 had a significant score for identifying deleterious nsSNPs. The importance of rs374941368 and rs200149541 in the prediction of post-translation changes was highlighted because it impacts a possible phosphorylation site. Gene-gene interactions revealed CCBE1's association with other genes, showing its role in a number of pathways and coexpressions. The top 16 deleterious nsSNPs found in this research should be investigated further in the future while researching diseases caused CCBE1 gene specifically HS. The FT web server predicted amino acid residues involved in the ligand-binding site of the CCBE1 protein, and two of the substitutions (R167W and T153N) were found to be involved. These highly deleterious nsSNPs can be used as marker pathogenic variants in the mutational diagnosis of the HS syndrome, and this research also offers potential insights that will aid in the development of precision medicines. CCBE1 proteins from Hennekam syndrome patients should be tested in animal models for this purpose. © 2021 Khyber Shinwari et al. The work was carried out within the framework of state research at the Institute of Immunology and Physiology of the Ural Branch of the Russian Academy of Sciences, project number AAAA-A21-121012090091-6.

Epigenetic regulation of cell and tissue function requires the coordinated action of transcription factors. However, their combinatorial activities during regeneration remain largely unexplored. Here, we discover an unexpected interaction between the cytoprotective transcription factor NRF2 and p63- a key player in epithelial morphogenesis. Chromatin immunoprecipitation combined with sequencing and reporter assays identifies enhancers and promoters that are simultaneously activated by NRF2 and p63 in human keratinocytes. Modeling of p63 and NRF2 binding to nucleosomal DNA suggests their chromatin-assisted interaction. Pharmacological and genetic activation of NRF2 increases NRF2–p63 binding to enhancers and promotes keratinocyte proliferation, which involves the common NRF2–p63 target cyclin-dependent kinase 12. These results unravel a collaborative function of NRF2 and p63 in the control of epidermal renewal and suggest their combined activation as a strategy to promote repair of human skin and other stratified epithelia.

Acute leukemias (ALs) of ambiguous lineage are a heterogeneous group of high-risk leukemias characterized by coexpression of myeloid and lymphoid markers. In this study, we identified a distinct subgroup of immature acute leukemias characterized by a broadly variable phenotype, covering acute myeloid leukemia (AML, M0 or M1), T/myeloid mixed-phenotype acute leukemia (T/M MPAL), and early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Rearrangements at 14q32/BCL11B are the cytogenetic hallmark of this entity. In our screening of 915 hematological malignancies, there were 202 AML and 333 T-cell acute lymphoblastic leukemias (T-ALL: 58, ETP; 178, non-ETP; 8, T/M MPAL; 89, not otherwise specified). We identified 20 cases of immature leukemias (4% of AML and 3.6% of T-ALL), harboring 4 types of 14q32/BCL11B translocations: t(2,14)(q22.3;q32) (n = 7), t(6;14)(q25.3;q32) (n = 9), t(7;14)(q21.2;q32) (n = 2), and t(8;14)(q24.2;q32) (n = 2). The t(2;14) produced a ZEB2-BCL11B fusion transcript, whereas the other 3 rearrangements displaced transcriptionally active enhancer sequences close to BCL11B without producing fusion genes. All translocations resulted in the activation of BCL11B, a regulator of T-cell differentiation associated with transcriptional corepressor complexes in mammalian cells. The expression of BCL11B behaved as a disease biomarker that was present at diagnosis, but not in remission. Deregulation of BCL11B co-occurred with variants at FLT3 and at epigenetic modulators, most frequently the DNMT3A, TET2, and/or WT1 genes. Transcriptome analysis identified a specific expression signature, with significant downregulation of BCL11B targets, and clearly separating BCL11B AL from AML, T-ALL, and ETP-ALL. Remarkably, an ex vivo drug-sensitivity profile identified a panel of compounds with effective antileukemic activity.

While the tumor suppressor protein p53 regulates the cell cycle to prevent cell damage, it also triggers apoptosis and prevents cancer. These inhibitory functions may disappear once the p53 gene is mutated. Under these circumstances, the detection of p53 protein concentrations can have significant clinical applications. In this study, nickel phthalocyanine (NiPc) was coated on a gold electrode to produce a modified Au/NiPc electrode. p53 antibodies were bonded to the Au/NiPc electrode by the Ni+2 ion in NiPc, which can be self-assembled with the imidazole group of the p53 protein. The Au/NiPc/anti-p53 electrode was subsequently dripped with a buffer solution of bovine serum albumin (BSA) to form the Au/NiPc/anti-p53/BSA electrode, which was used for the detection of p53 antigen under 10 mM potassium ferricyanide/potassium ferrocyanide (K3Fe(CN)6/K4Fe(CN)6) solution by cyclic voltammetry and differential pulse voltammetry analyses. The linear detection range and the sensitivity for the p53 antigen were 0.1&ndash, 500 pg/mL and 60.65 &mu, A/Log (pg/mL)-cm2, respectively, with a detection time of 90&ndash, 150 s. In addition, Au/NiPc/anti-p53 (100 ng/mL)/BSA electrodes were tested for specificity using glucose, bovine serum albumin, histidine, ascorbic acid, uric acid, prostate-specific antigen, human serum albumin, and human immunoglobulin G. All p-values were &lt, 0.0005, indicating an outstanding specificity.

© 2020 by the authors., HERC proteins are ubiquitin E3 ligases of the HECT family. The HERC subfamily is composed of six members classified by size into large (HERC1 and HERC2) and small (HERC3–HERC6). HERC family ubiquitin ligases regulate important cellular processes, such as neurodevelopment, DNA damage response, cell proliferation, cell migration, and immune responses. Accumulating evidence also shows that this family plays critical roles in cancer. In this review, we provide an integrated view of the role of these ligases in cancer, highlighting their bivalent functions as either oncogenes or tumor suppressors, depending on the tumor type. We include a discussion of both the molecular mechanisms involved and the potential therapeutic strategies., J.L.R. is supported by the Spanish Ministry of Science and Innovation (BFU2016-80295-R, SAF2017-90900-REDT, and UBIRed). X.R.B. is supported by grants from the Castilla-León Government (CSI252P18, CLC-2017-01), the Spanish Ministry of Science and Innovation (SAF2015-64556-R, RTI2018-096481-B-100) and the Spanish Association against Cancer (GC16173472GARC). X.R.B.’s institution is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Castilla-León autonomous government (CLC-2017-01). Both Spanish and Castilla-León government-associated funding is partially supported by the European Regional Development Fund. J.S.-G. and A.M.-M. received FPU Fellowships (FPU17/02413 and FPU18/06325, respectively) from the Spanish Ministry of Science, Innovation and Universities.

Unmethylated O6-methylguanine-DNA-methyltransferase (MGMT) promoter leads to Temozolomide (TMZ) resistance in most of the glioblastoma multiforme (GBM) patients. We previously investigated the synergistic effect of Olea europaea leaf extract (OLE) on TMZ cytotoxicity through modulating microRNA expression. To date, knowledge about the effect of OLE on MGMT methylation is insufficient. The aim of the current study was to evaluate the potential modulating effect of OLE on the TMZ response of GBM tumors through MGMT methylation. Exposure to 1mg/mL OLE caused a significant induction of CpG island methylation in the MGMT gene using Methyl quantitative PCR assay (P < 0.001). In WST-1 analysis, the use of 350 mu M TMZ plus 1mg/mL OLE significantly increased the TMZ response of MGMT unmethylated cells (P = 0.003). Using the comet assay, the impact of 1mg/mL OLE plus 350 mu M TMZ on the formation of DNA strand breaks was significantly higher than that of 450 mu M TMZ alone (P < 0.001) and Western blot analysis revealed that, when cells are treated with 1-mg/mL OLE, the total p53 protein levels tended to decrease. The results presented in this study uniquely demonstrated that OLE synergistically increased the TMZ response of GBM tumors by regulating MGMT gene methylation and p53 expression. However, further studies to validate our findings are required.

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

The p53 tumor suppressor protein is a transcription factor that plays a prominent role in protecting cells from malignant transformation. Protein levels of p53 and its transcriptional activity are tightly regulated by the ubiquitin E3 ligase MDM2, the gene expression of which is transcriptionally regulated by p53 in a negative feedback loop. The p53 protein is transcriptionally active as a tetramer, and this oligomerization state is modulated by a complex formed by NEURL4 and the ubiquitin E3 ligase HERC2. Here, we report that MDM2 forms a complex with oligomeric p53, HERC2, and NEURL4. HERC2 knockdown results in a decline in MDM2 protein levels without affecting its protein stability, as it reduces its mRNA expression by inhibition of its promoter activation. DNA damage induced by bleomycin dissociates MDM2 from the p53/HERC2/NEURL4 complex and increases the phosphorylation and acetylation of oligomeric p53 bound to HERC2 and NEURL4. Moreover, the MDM2 promoter, which contains p53‐response elements, competes with HERC2 for binding of oligomeric, phosphorylated and acetylated p53. We integrate these findings in a model showing the pivotal role of HERC2 in p53‐MDM2 loop regulation. Altogether, these new insights in p53 pathway regulation are of great interest in cancer and may provide new therapeutic targets., The ubiquitin ligase MDM2 regulates protein levels of the p53 tumor suppressor protein. p53 is a transcription factor that regulates MDM2 expression. This study reports the formation of a NEURL4/HERC2/oligomeric p53/MDM2 complex and how it participates in the stability of p53 and MDM2 proteins and in the transcriptional activation of p53 in response to DNA damage.

Ubiquitin ligases regulate numerous cellular processes, including tissue homeostasis, cellular metabolism, and cell cycle progression. These enzymes recognize, interact with and ubiquitylate specific substrates. Homologous to the E6-AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing proteins (HERCs) belong to the family of HECT ubiquitin ligases. There are six human HERCs which can be divided into two subgroups: large HERCs (HERC1-2) and small HERCs (HERC3-6). Alterations in the function of large HERCs are associated with serious pathologies such as neurological disorders. Mutations in human HERC1 have been associated with overgrowth, intellectual disability and some autistic features; while mutations in HERC2 have been identified as the cause of a neurodevelopmental disorder with similarities to Angelman syndrome and autism.

Malformations of the human cortex represent a major cause of disability1. Mouse models with mutations in known causal genes only partially recapitulate the phenotypes and are therefore not unlimitedly suited for understanding the molecular and cellular mechanisms responsible for these conditions(2). Here we study periventricular heterotopia (PH) by analyzing cerebral organoids derived from induced pluripotent stem cells (iPSCs) of patients with mutations in the cadherin receptor-ligand pair DCHS1 and FAT4 or from isogenic knockout (KO) lines(1,3). Our results show that human cerebral organoids reproduce the cortical heterotopia associated with PH. Mutations in DCHS1 and FAT4 or knockdown of their expression causes changes in the morphology of neural progenitor cells and result in defective neuronal migration dynamics only in a subset of neurons. Single-cell RNA-sequencing (scRNA-seq) data reveal a subpopulation of mutant neurons with dysregulated genes involved in axon guidance, neuronal migration and patterning. We suggest that defective neural progenitor cell (NPC) morphology and an altered navigation system in a subset of neurons underlie this form of PH.

Background We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme alpha-enolase (ENO1) also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. Methods The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1) PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM). The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. Results We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN) via urokinase plasminogen activator receptor (uPAR). Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell migration and invasion of shENO1 PDA cells was observed. Conclusion These data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0385-8) contains supplementary material, which is available to authorized users.

The PALB2 gene, has been accepted as a moderate-penetrance gene associated with breast cancer susceptibility and this gene product is involved in the DNA damage repair pathway via co-localization with BRCA2. Germline PALB2 mutations are associated with an increased breast cancer risk. However, the prevalence of the diverse types of PALB2 variants depend on the population. Thus, the aim of the present study was to determine, for the first time, the prevalence of PALB2 variants in a Turkish population of BRCA1/BRCA2-negative early-onset patients with breast cancer. In total, 223 Turkish patients with BRCA1/BRCA2 negative early-onset breast cancer and 60 unaffected women were included in the study. All the coding exons and intron/exon boundaries of PALB2 were subjected to mutational analysis by heteroduplex analysis (HDA) and DNA sequencing. Eighteen PALB2 variants were found in breast cancer patients within the Turkish population. Three variants (c.271G>A, c.404C>A and c.2981T>A) have not been previously reported. In addition, nine intronic variants were described, and this study is the first to describe the c.1685-44T>A intronic variant. The prevalence of possible pathogenic PALB2 variants was found to be 4.03 % in BRCA1/2-negative Turkish patients with early-onset breast cancer. Different variants of PALB2 have been reported in the literature, and the prevalence of these variants could different for each population. This is the first study to investigate the prevalence of PALB2 variants in Turkish patients with early-onset breast cancer. British Association for Psychopharmacology

Aim MYC deregulation contributes to breast cancer development and progression. Deregulated expression levels of long non-coding RNAs (lncRNA) have been demonstrated to be critical players in development and/or maintenance of breast cancer. In this study we aimed to evaluate lncRNA expressions depending on MYC overexpression and knockdown in breast cancer cells. Materials and methods Cells were infected with lentiviral vectors by either knockdown or overexpression of c-MYC. LncRNA cDNA was transcribed from total RNA samples and lncRNAs were evaluated by qRT-PCR. Results Our results indicated that some of the lncRNAs having tumor suppressor (GAS5, MEG3, lincRNA-p21) and oncogenic roles (HOTAIR) are regulated by c-MYC. Conclusion We observed that c-MYC regulates lncRNAs that have important roles on proliferation, cell cycle and etc. Further studies will give us a light to identify molecular mechanisms related to MYC-lncRNA regulatory pathways in breast cancer.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Cancer research has been heavily geared towards genomic events in the development and progression of cancer. In contrast, metabolic regulation, such as aberrant metabolism in cancer, is poorly understood. Alteration in cellular metabolism was once regarded simply as a consequence of cancer rather than as playing a primary role in cancer promotion and maintenance. Resurgence of cancer metabolism research has identified critical metabolic reprogramming events within biosynthetic and bioenergetic pathways needed to fulfill the requirements of cancer cell growth and maintenance. The tumor suppressor protein p53 is emerging as a key regulator of metabolic processes and metabolic reprogramming in cancer cells—balancing the pendulum between cell death and survival. This review provides an overview of the classical and emerging non-classical tumor suppressor roles of p53 in regulating mitochondrial dynamics: mitochondrial engagement in cell death processes in the prevention of cancer. On the other hand, we discuss p53 as a key metabolic switch in cellular function and survival. The focus is then on the conceivable roles of p53 in breast cancer metabolism. Understanding the metabolic functions of p53 within breast cancer metabolism will, in due course, reveal critical metabolic hotspots that cancers advantageously re-engineer for sustenance. Illustration of these events will pave the way for finding novel therapeutics that target cancer metabolism and serve to overcome the breast cancer burden.

Epigenetic mechanisms are involved in gastrointestinal (GI) cancer pathogenesis. Insights into the molecular basis of GI carcinogenesis led to the identification of different epigenetic pathways and signatures that may play a role as therapeutic targets in metastatic colorectal cancer (mCRC) and non-colorectal GI tumors. Among these alterations, O6-methylguanine DNA methyltransferase (MGMT) gene promoter methylation is the most investigated biomarker and seems to be an early and frequent event, at least in CRC. Loss of expression of MGMT as a result of gene promoter methylation has been associated with interesting activity of alkylating agents in mCRC. However, the optimal methods for the definition of the MGMT status and additional predictive factors beyond MGMT in GI malignancies are lacking. Here we review the current role of MGMT methylation and other epigenetic alterations as potential treatment targets in GI tumors.

Metastasis is respoMetastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors.

Tumors with histological features of pilocytic astrocytoma (PA), but with increased mitotic activity and additional high-grade features (particularly microvascular proliferation and palisading necrosis) have often been designated anaplastic pilocytic astrocytomas. The status of these tumors as a separate entity has not yet been conclusively demonstrated and molecular features have only been partially characterized. We performed DNA methylation profiling of 102 histologically defined anaplastic pilocytic astrocytomas. T-distributed stochastic neighbor-embedding(t-SNE) and hierarchical clustering analysis of these 102 cases against 158 reference cases from 12 glioma reference classes revealed that a subset of 83 of these tumors share a common DNA methylation profile that is distinct from the reference classes. These 83 tumors were thus denominated DNA methylation class anaplastic astrocytoma with piloid features (MC AAP). The 19 remaining tumors were distributed amongst the reference classes, with additional testing confirming the molecular diagnosis in most cases. Median age of patients with MC AAP was 41.5years. The most frequent localization was the posterior fossa (74%). Deletions of CDKN2A/B (66/83, 80%), MAPK pathway gene alterations (49/65, 75%, most frequently affecting NF1, followed by BRAF and FGFR1) and mutations of ATRX or loss of ATRX expression (33/74, 45%) were the most common molecular alterations. All tumors were IDH1/2 wildtype. The MGMT promoter was methylated in 38/83 tumors (45%). Outcome analysis confirmed an unfavorable clinical course in comparison to PA, but better than IDH wildtype glioblastoma. In conclusion, we show that a subset of histologically defined anaplastic pilocytic astrocytomas forms a separate DNA methylation cluster, harbors recurrent alterations in MAPK pathway genes in combination with alterations of CDKN2A/B and ATRX, affects patients who are on average older than those diagnosed with PA and has an intermediate clinical outcome.

Hepatocellular carcinoma (HCC) is a heterogeneous tumour associated with poor prognostic outcome. Caveolin-1 (CAV1), a membrane protein involved in the formation of caveolae, is frequently overexpressed in HCC. Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine having a dual role in hepatocarcinogenesis: inducer of apoptosis at early phases, but pro-tumourigenic once cells acquire mechanisms to overcome its suppressor effects. Apoptosis induced by TGF-β is mediated by upregulation of the NADPH oxidase NOX4, but counteracted by transactivation of the epidermal growth factor receptor (EGFR) pathway. Previous data suggested that CAV1 is required for the anti-apoptotic signals triggered by TGF-β in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-β response in HCC cell lines that express different levels of CAV1. Accordingly, stable CAV1 knockdown or overexpressing cell lines were generated. We demonstrate that CAV1 is protecting HCC cells from TGF-β-induced apoptosis, which attenuates its suppressive effect on clonogenic growth and increases its effects on cell migration. Downregulation of CAV1 in HLE cells promotes TGF-β-mediated induction of the pro-apoptotic BMF, which correlates with upregulation of NOX4, whereas CAV1 overexpression in Huh7 cells shows the opposite effect. CAV1 silenced HLE cells show attenuation in TGF-β-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-β activating the EGFR pathway, acquire the capacity to do so when CAV1 is overexpressed. Analyses in samples from HCC patients revealed that tumour tissues presented higher expression levels of CAV1 compared with surrounding non-tumoural areas. Furthermore, a significant positive correlation among the expression of CAV1 and TGFB1 was observed. We conclude that CAV1 has an essential role in switching the response to TGF-β from cytostatic to tumourigenic, which could have clinical meaning in patient stratification.

SUMMARY Renal cell carcinoma (RCC) is not a single disease, but several histologically defined cancers with different genetic drivers, clinical courses, and therapeutic responses. The current study evaluated 843 RCC from the three major histologic subtypes, including 488 clear cell RCC, 274 papillary RCC, and 81 chromophobe RCC. Comprehensive genomic and phenotypic analysis of the RCC subtypes reveals distinctive features of each subtype that provide the foundation for the development of subtype-specific therapeutic and management strategies for patients affected with these cancers. Somatic alteration of BAP1, PBRM1, and PTEN and altered metabolic pathways correlated with subtype-specific decreased survival, while CDKN2A alteration, increased DNA hypermethylation, and increases in the immune-related Th2 gene expression signature correlated with decreased survival within all major histologic subtypes. CIMP-RCC demonstrated an increased immune signature, and a uniform and distinct metabolic expression pattern identified a subset of metabolically divergent (MD) ChRCC that associated with extremely poor survival., Graphical abstract In Brief Ricketts et al. find distinctive features of each RCC subtype, providing the foundation for development of subtypespecific therapeutic and management strategies. Somatic alteration of BAP1, PBRM1, and metabolic pathways correlates with subtype-specific decreased survival, while CDKN2A alteration, DNA hypermethylation, and Th2 immune signature correlate with decreased survival within all subtypes.

ΔNp63α is finely and strictly regulated during embryogenesis and differentiation. ΔNp63α is the only p63 isoform degraded by the proteasome after Ubiquitin and SUMO (Small Ubiquitin-like MOdifier) conjugation. Here, we show that p63 ubiquitylation per se is not the signal triggering p63 proteasomal degradation. Taking advantage of natural ΔNp63α mutants isolated by patients with Split Hand and Foot Malformation IV syndrome, we found that SUMO and Ub modifications are not redundant and both are required to guarantee efficient ΔNp63α degradation. Here, we present evidence that sumoylation and ubiquitylation of ΔNp63α are strongly intertwined, and none of the two can efficiently occur if the other is impaired.

The YAP and TAZ mediators of the Hippo pathway (hereafter called YAP/TAZ) promote tissue proliferation and organ growth. However, how their biological properties intersect with cellular metabolism remains unexplained. Here, we show that YAP/TAZ activity is controlled by the SREBP/mevalonate pathway. Inhibition of the rate-limiting enzyme of this pathway (HMG-CoA reductase) by statins opposes YAP/TAZ nuclear localization and transcriptional responses. Mechanistically, the geranylgeranyl pyrophosphate produced by the mevalonate cascade is required for activation of Rho GTPases that, in turn, activate YAP/TAZ by inhibiting their phosphorylation and promoting their nuclear accumulation. The mevalonate-YAP/TAZ axis is required for proliferation and self-renewal of breast cancer cells. In Drosophila melanogaster, inhibition of mevalonate biosynthesis and geranylgeranylation blunts the eye overgrowth induced by Yorkie, the YAP/TAZ orthologue. In tumour cells, YAP/TAZ activation is promoted by increased levels of mevalonic acid produced by SREBP transcriptional activity, which is induced by its oncogenic cofactor mutant p53. These findings reveal an additional layer of YAP/TAZ regulation by metabolic cues.

Protein phosphorylation is a key mechanism by which normal and cancer cells regulate their main transduction pathways. Protein kinases and phosphatases are precisely orchestrated to achieve the (de)phosphorylation of candidate proteins. Indeed, cellular health is dependent on the fine-tune of phosphorylation systems, which when deregulated lead to cancer. Transforming growth factor beta (TGF-β) pathway involvement in the genesis of prostate cancer has long been established. Many of its members were shown to be hypo- or hyperphosphorylated during the process of malignancy. A major phosphatase that is responsible for the vast majority of the serine/threonine dephosphorylation is the phosphoprotein phosphatase 1 (PPP1). PPP1 has been associated with the dephosphorylation of several proteins involved in the TGF-β cascade. This review will discuss the role of PPP1 in the regulation of several TGF-β signalling members and how the subversion of this pathway is related to prostate cancer development. Furthermore, current challenges on the protein phosphatases field as new targets to cancer therapy will be addressed. published

One of the hallmarks of Alzheimer's disease (AD) is the accumulation and deposition of amyloid-β (Aβ) peptides in the brain and cerebral vasculature. Aβ evokes neuroinflammation and has been implicated in insulin signaling disruption and JNK-AP1 activation, contributing to AD neuropathologies including oxidative injury and vascular insufficiencies. In this study we aim to better understand the protective mechanisms of insulin signaling and JNK-AP1 inhibition on the adverse effects of Aβ. Four-hour treatment of hCMEC/D3, the immortalized human brain endothelial cells (iHBEC), with Aβ1-42 resulted in significant c-Jun phosphorylation, oxidative stress, and cell toxicity. Concurrent treatment with Aβ1-42 and insulin or Aβ1-42 and JNK inhibitor SP600125 significantly improved cell viability. Cytokine array on conditioned media showed that insulin and SP600125 strongly reduced all Aβ 1-42-induced cytokines. ELISA confirmed the protective effect of insulin and SP600125 on Aβ-induced expression of interleukin (IL)-8 and Growth related oncogene-α (Gro-α). qRT-PCR revealed that insulin and SP600125 protected iHBEC from Aβ1-42-induced inflammatory gene expression. Transcription factor profiling showed that treatment of iHBEC with Aβ1-42, insulin, or SP600125 alone or in combination resulted in profound changes in modulating the activities of multiple transcription factors and relevant pathways, some of which were validated by western blot. Insulin treatment and JNK inhibition in vitro synergistically reduced c-Jun phosphorylation and thus JNK-AP1 signaling activation. The study suggests that activation of insulin and blocking of JNK-AP1 signaling inhibits Aβ-induced dysregulation of insulin signaling and inflammatory response.

Complex phenotypes emerge from the interactions of thousands of macromolecules that are organized in multimolecular complexes and interacting functional modules. In turn, modules form functional networks in health and disease. Omics approaches collect data on changes for all genes and proteins and statistical analysis attempts to uncover the functional modules that perform the functions that characterize higher levels of biological organization. Systems biology attempts to transcend the study of individual genes/proteins and to integrate them into higher order information. Cancer cells exhibit defective genetic and epigenetic networks formed by altered complexes and network modules arising in different parts of tumor tissues that sustain autonomous cell behavior which ultimately lead tumor growth. We suggest that an understanding of tumor behavior must address not only molecular but also, and more importantly, tumor cell heterogeneity, by considering cancer tissue genetic and epigenetic networks, by characterizing changes in the types, composition, and interactions of complexes and networks in the different parts of tumor tissues, and by identifying critical hubs that connect them in time and space.

BRCA1-associated protein 1 (BAP1) is a potent tumour suppressor gene that modulates environmental carcinogenesis. All carriers of inherited heterozygous germline BAP1-inactivating mutations (BAP1 +/-) developed one and often several BAP1 -/- malignancies in their lifetime, mostly malignant mesothelioma, uveal melanoma, and so on. Moreover, BAP1-acquired biallelic mutations are frequent in human cancers. BAP1 tumour suppressor activity has been attributed to its nuclear localization, where it helps to maintain genome integrity. The possible activity of BAP1 in the cytoplasm is unknown. Cells with reduced levels of BAP1 exhibit chromosomal abnormalities and decreased DNA repair by homologous recombination, indicating that BAP1 dosage is critical. Cells with extensive DNA damage should die and not grow into malignancies. Here we discover that BAP1 localizes at the endoplasmic reticulum. Here, it binds, deubiquitylates, and stabilizes type 3 inositol-1,4,5-trisphosphate receptor (IP3R3), modulating calcium (Ca 2+) release from the endoplasmic reticulum into the cytosol and mitochondria, promoting apoptosis. Reduced levels of BAP1 in BAP1 +/- carriers cause reduction both of IP3R3 levels and of Ca 2+ flux, preventing BAP1 +/- cells that accumulate DNA damage from executing apoptosis. A higher fraction of cells exposed to either ionizing or ultraviolet radiation, or to asbestos, survive genotoxic stress, resulting in a higher rate of cellular transformation. We propose that the high incidence of cancers in BAP1 +/- carriers results from the combined reduced nuclear and cytoplasmic activities of BAP1. Our data provide a mechanistic rationale for the powerful ability of BAP1 to regulate gene-environment interaction in human carcinogenesis.

Background: Germline mutations of the oncosuppressor gene breast cancer 1-associated protein 1 (BAP1) were recently related to an autosomal-dominant tumor predisposition syndrome (BAP1-TPDS), characterized by uveal melanoma, malignant mesothelioma (MM), cutaneous melanoma, and other malignancies. The demonstration that BAP1 mutations are strongly associated with MM has provided a real breakthrough in the study of genetic predisposition in MM, that may explain why only a fraction of asbestos-exposed individuals go on to develop MM. Materials and Methods: To evaluate the possible role of BAP1 mutations in the epidemiology of sporadic MM, and their relationship with asbestos exposure, we determined the prevalence of germline BAP1 mutations by the Sanger method in a group of 29 asbestos-exposed patients, 21 of which were diagnosed with MM. They were residents of Trieste, a ship-building town in Northeast Italy with a very high incidence of mesothelioma. Results: We identified non-obviously pathogenetic germline sequence variants of BAP1 in 3/29 patients and in 2/21 MM cases (10%). Conclusion: Non obviously pathogenic germline sequence variants of BAP1 were found. Nevertheless, limitations of predictive web tools allowed us to comment on some interesting peculiarities of our findings.

Left ventricular (LV) remodelling after myocardial infarction (MI) is a crucial determinant of the clinical course of heart failure. Matrix metallo-proteinase (MMP) activation is strongly associated with LV remodelling after MI. Elucidation of plasma membrane receptors related to the acti-vation of specific MMPs is fundamental for treating adverse cardiac remodelling after MI. The aim of current investigation was to explore thepotential association between the low-density lipoprotein receptor-related protein 1 (LRP1) and MMP-9 and MMP-2 spatiotemporal expressionafter MI. Real-time PCR and Western blot analyses showed that LRP1 mRNA and protein expression levels, respectively, were significantlyincreased in peri-infarct and infarct zones at 10 and 21 days after MI. Confocal microscopy demonstrated high colocalization between LRP1and the fibroblast marker vimentin, indicating that LRP1 is mostly expressed by cardiac fibroblasts in peri-infarct and infarct areas. LRP1 alsocolocalized with proline-rich tyrosine kinase 2 (pPyk2) and MMP-9 in cardiac fibroblasts in ischaemic areas at 10 and 21 days after MI. Cell cul-ture experiments revealed that hypoxia increases LRP1, pPyk2 protein levels and MMP-9 activity in fibroblasts, without significant changes inMMP-2 activity. MMP-9 activation by hypoxia requires LRP1 and Pyk2 phosphorylation in fibroblasts. Collectively, ourin vivoandin vitrodatasupport a major role of cardiac fibroblast LRP1 levels on MMP-9 up-regulation associated with ventricular remodelling after MI, The research leading to these results received funding from the Instituto de Salud Carlos III (FIS PI14/01729 and FIS PI14/01682) that was cofinanced by the European Fund for Regional Development, as well as from the Ministerio de Ciencia e Innovaci on (SAF2011-30067-C02-01), the Fundacio La Marato de TV3 (201521_10, 201502_30 and 080330), the Red de Terapia Celular-TerCel (RD12/0019/0029), the Red Cardio-vascular (RD12/0042/0027 and RD12/ 0042/0047), the Sociedad Espa~nola de Cardiolog ıa and the Fundaci o Privada Daniel Bravo Andreu. E.R.L. is a recipient of an FI-DGR_B01008 grant from Generalitat de Catalunya, and D.G. is a recipient of CD14/0109 from Sara Borell of the Instituto de Salud Carlos III.

Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with β-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants., Summary: Transcripts of the tumor suppressor gene SHREW-1 exist in various splice variants in human and mouse encoding proteins with a differential expression and intracellular localization profile.

Aberrant methylation of promoter CpG islands is known to be a major inactivation mechanism of the tumor-related genes including DNA repair genes. The objective of this study was to determine the frequency of promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene as a DNA repair gene in nonsmall cell lung cancer (NSCLC) and to analyze the correlation with clinicopathological parameters including age, gender, smoking status, histological subtype, and clinical stage. Eighty patients with NSCLC were included in this study. The analysis of DNA methylation was performed on formalin-fixed, paraffin-embedded lung cancer tissues. Following DNA isolation and bisulfite treatment, DNA methylation was analyzed by methylation-specific real-time polymerase chain reaction. MGMT promoter methylation was detected in 51 of 80 (64%) NSCLC patients. There was a significant correlation between MGMT methylation and tumor stage (p=0.01). The frequencies of the promoter methylation of MGMT gene in smokers and older patients were higher than in their counterparts. In conclusion, the present study provides strong evidence for a higher frequency of promoter methylation of the MGMT gene in NSCLC, indicating that it is a common event during the carcinogenesis of NSCLC. © 2011 Mary Ann Liebert, Inc.

Summary Autophagy, a major degradation process for long-lived and aggregate-prone proteins, affects various human processes, such as development, immunity, cancer, and neurodegeneration. Several autophagy regulators have been identified in recent years. Here we show that nitric oxide (NO), a potent cellular messenger, inhibits autophagosome synthesis via a number of mechanisms. NO impairs autophagy by inhibiting the activity of S-nitrosylation substrates, JNK1 and IKKβ. Inhibition of JNK1 by NO reduces Bcl-2 phosphorylation and increases the Bcl-2–Beclin 1 interaction, thereby disrupting hVps34/Beclin 1 complex formation. Additionally, NO inhibits IKKβ and reduces AMPK phosphorylation, leading to mTORC1 activation via TSC2. Overexpression of nNOS, iNOS, or eNOS impairs autophagosome formation primarily via the JNK1–Bcl-2 pathway. Conversely, NOS inhibition enhances the clearance of autophagic substrates and reduces neurodegeneration in models of Huntington's disease. Our data suggest that nitrosative stress-mediated protein aggregation in neurodegenerative diseases may be, in part, due to autophagy inhibition., Graphical Abstract Highlights ► NO inhibits autophagy by independently inhibiting JNK1 and IKKβ ► NO inhibits autophagic flux via mTOR and mTOR-independent routes ► NOS overexpression impairs autophagosome synthesis via JNK1–Bcl-2 pathway ► NOS inhibition induces autophagy and protects against neurodegeneration

The Ink4/Arf tumour suppressor locus encodes three potent tumour suppressors, namely p16Ink4a, p15Ink4b and p19Arf. Here Li et al. show that the locus is rate limiting for reprogramming, and its transient inhibition significantly improves the generation of iPS cells. The also show ageing upregulates the Ink4/Arf locus and, accordingly, reprogramming is less efficient in cells from old organisms. The Ink4/Arf tumour suppressor locus encodes the three potent tumour suppressors p16Ink4a, p15Ink4b and p19Arf. Here the locus is shown to be rate-limiting for reprogramming, and its transient inhibition significantly improves the generation of induced pluripotent stem cells. Furthermore, ageing is shown to upregulate the Ink4/Arf locus, with less efficient reprogramming seen in cells from old organisms. The mechanisms involved in the reprogramming of differentiated cells into induced pluripotent stem (iPS) cells by the three transcription factors Oct4 (also known as Pou5f1), Klf4 and Sox2 remain poorly understood1. The Ink4/Arf locus comprises the Cdkn2a–Cdkn2b genes encoding three potent tumour suppressors, namely p16Ink4a, p19Arf and p15Ink4b, which are basally expressed in differentiated cells and upregulated by aberrant mitogenic signals2,3,4. Here we show that the locus is completely silenced in iPS cells, as well as in embryonic stem (ES) cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated after differentiation. Cell culture conditions during reprogramming enhance the expression of the Ink4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, the three factors together repress the Ink4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of ‘stemness’. This downregulation also occurs in cells carrying the oncoprotein large-T, which functionally inactivates the pathways regulated by the Ink4/Arf locus, thus indicating that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of the Ink4/Arf locus has a profound positive effect on the efficiency of iPS cell generation, increasing both the kinetics of reprogramming and the number of emerging iPS cell colonies. In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming by activation of p53 (encoded by Trp53) and p21 (encoded by Cdkn1a); whereas, in human fibroblasts, INK4a is more important than ARF. Furthermore, organismal ageing upregulates the Ink4/Arf locus2,5 and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with a short hairpin RNA. All together, we conclude that the silencing of Ink4/Arf locus is rate-limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS cells.

Nuclear exclusion of the PTEN (phosphatase and tensin homologue deleted in chromosome 10) tumour suppressor has been associated with cancer progression. However, the mechanisms leading to this aberrant PTEN localization in human cancers are currently unknown. We have previously reported that ubiquitinylation of PTEN at specific lysine residues regulates its nuclear-cytoplasmic partitioning. Here we show that functional promyelocytic leukaemia protein (PML) nuclear bodies co-ordinate PTEN localization by opposing the action of a previously unknown PTEN-deubiquitinylating enzyme, herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), and that the integrity of this molecular framework is required for PTEN to be able to enter the nucleus. We find that PTEN is aberrantly localized in acute promyelocytic leukaemia, in which PML function is disrupted by the PML-RARalpha fusion oncoprotein. Remarkably, treatment with drugs that trigger PML-RARalpha degradation, such as all-trans retinoic acid or arsenic trioxide, restore nuclear PTEN. We demonstrate that PML opposes the activity of HAUSP towards PTEN through a mechanism involving the adaptor protein DAXX (death domain-associated protein). In support of this paradigm, we show that HAUSP is overexpressed in human prostate cancer and is associated with PTEN nuclear exclusion. Thus, our results delineate a previously unknown PML-DAXX-HAUSP molecular network controlling PTEN deubiquitinylation and trafficking, which is perturbed by oncogenic cues in human cancer, in turn defining a new deubiquitinylation-dependent model for PTEN subcellular compartmentalization.

Initiation, progression and evasion are sequential steps in cancer formation, with autonomous cell proliferation as a final outcome. Genetic or epigenetic alterations of key regulatory genes of the cell cycle are frequently associated with these phenomena. Recently, chromosomal instability, a long-supposed driving force of tumorigenesis, was associated with dysregulation of mitotic genes, providing advantages to tumor cells. Numerous molecules thus provide a key link in the chain of relationships between chromosomal instability and cancer. Here, we discuss emerging evidence revealing that two p53 family members, p53 and p73, might be key regulatory genes at the heart of the relationship between chromosomal instability and cancer. We argue that the role of members of the p53 family as tumor suppressor proteins, their impact on the control of cellular ploidy, and their newly emerging connection with mitotic checkpoint regulatory genes support the suggestion that p73 and p53 could be two of the missing links among chromosomal instability, the mitotic checkpoint and cancer.

Sayar Atasoy, Nilufer/0000-0002-3247-613X WOS: 000379274300004 PubMed: 27215390 Cutaneous squamous cell carcinomas (cSCCs) are common human carcinomas. Despite having metastasizing capacities, they usually show less aggressive progression compared to squamous cell carcinoma (SCC) of other organs. Metastasis suppressor proteins (MSPs) are a group of proteins that control and slow-down the metastatic process. In this study, we established the importance of seven well-defined MSPs including NDRG1, NM23-H1, RhoGDI2, E-cadherin, CD82/KAI1, MKK4, and AKAP12 in cSCCs. Protein expression levels of the selected MSPs were detected in 32 cSCCs, 6 in situ SCCs, and two skin cell lines (HaCaT, A-431) by immunohistochemistry. The results were evaluated semi-quantitatively using the HSCORE system. In addition, mRNA expression levels were detected by qRT-PCR in the cell lines. The HSCOREs of NM23-H1 were similar in cSCCs and normal skin tissues, while RGHOGDI2, E-cadherin and AKAP12 were significantly downregulated in cSCCs compared to normal skin. The levels of MKK4, NDRG1 and CD82 were partially conserved in cSCCs. In stage I SCCs, nuclear staining of NM23-H1 (NM23-H1nuc) was significantly lower than in stage SCCs. Only nuclear staining of MKK4 (MKK4nuc) showed significantly higher scores in in situ carcinomas compared to invasive SCCs. In conclusion, similar to other human tumors, we have demonstrated complex differential expression patterns for the MSPs in in-situ and invasive cSCCs. This complex MSP signature warrants further biological and experimental pathway research. (C) 2016 Elsevier GmbH. All rights reserved. Scientific and Technical Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [SBAG-108S184] This study was financially supported by the Scientific and Technical Research Council of Turkey (TUBITAK, grant number SBAG-108S184). The project was approved by the Kirikkale University Local Ethics Committee (07.04.2008/2008-039).

Alpha-enolase is a key glycolytic enzyme that plays a functional role in several physiological processes depending on the cellular localization. The enzyme is mainly localized in the cytoplasm whereas an alternative translated form, named MBP-1, is predominantly nuclear. The MBP-1 protein has been characterized as a c-Myc promoter binding protein that negatively controls transcription. In the present study, we identified the kelch protein NS1-BP as one of the alpha-enolase/MBP-1 partners by using a yeast two-hybrid screening. Although NS1-BP has been originally described as a protein mainly localized in the nucleus, we provide evidence that NS1-BP also interacts with actin in human cells, as reported for most kelch-containing proteins. Here we showed that alpha-enolase and MBP-1 associate with NS1-BP in vitro and in vivo by GST pull-down assays and coimmunoprecipitation experiments; subsequent immunofluorescent staining confirmed colocalization of the proteins within the cells. Furthermore, functional analyses performed by cotransfection assays revealed that NS1-BP enhances the inhibitory effect exerted by MBP-1 on c- Myc promoter. In mammalian cells, the overexpression of both proteins resulted in an increased repression of basal c-Myc transcription and consistently affected the steady state levels of endogenous c-Myc mRNA. These findings further support the distinct roles of alpha-enolase and its MBP-1 variant in maintaining cell homeostasis. Moreover, our data suggest a novel function for NS1-BP in the control of cell proliferation. © 2007 Elsevier B.V. All rights reserved.

The balance between antiapoptotic and proapoptotic proteins of the Bcl-2 family is critical in determining the fate of T cells in response to death stimuli. Proapoptotic genes, such as bax, are generally regulated by the p53 family of transcription factors, whereas NF-kappaB subunits can activate the transcription of antiapoptotic Bcl-2 members. Here, we show that CD28 activation protects memory T cells from irradiation-induced apoptosis by both upregulating bcl-xL and inhibiting bax gene expression. We found that p73, but not p53, binds to and trans-activates the bax gene promoter in irradiated T cells. The activation of RelA/NF-kappaB subunit in CD28 costimulated T cells and its binding onto the bax gene promoter results in suppression of bax transcription and decrease in both p73 and RNA polymerase II recruitment in vivo. RelA recruitment on the bax gene promoter is also accompanied by the lost of p300 binding and the parallel appearance of histone deacetylase-1-containing complexes. These findings identify RelA/NF-kappaB as a critical regulator of T-cell survival by affecting the balance of Bcl-2 family members.

Introduction Basal-like breast cancer (BLBC) is an aggressive subtype often characterized by distant metastasis, poor patient prognosis, and limited treatment options. Therefore, the discovery of alternative targets to restrain its metastatic potential is urgently needed. In this study, we aimed to identify novel genes that drive metastasis of BLBC and to elucidate the underlying mechanisms of action. Methods An unbiased approach using gene expression profiling of a BLBC progression model and in silico leveraging of pre-existing tumor transcriptomes were used to uncover metastasis-promoting genes. Lentiviral-mediated knockdown of interleukin-13 receptor alpha 2 (IL13Ralpha2) coupled with whole-body in vivo bioluminescence imaging was performed to assess its role in regulating breast cancer tumor growth and lung metastasis. Gene expression microarray analysis was followed by in vitro validation and cell migration assays to elucidate the downstream molecular pathways involved in this process. Results We found that overexpression of the decoy receptor IL13Ralpha2 is significantly enriched in basal compared with luminal primary breast tumors as well as in a subset of metastatic basal-B breast cancer cells. Importantly, breast cancer patients with high-grade tumors and increased IL13Ralpha2 levels had significantly worse prognosis for metastasis-free survival compared with patients with low expression. Depletion of IL13Ralpha2 in metastatic breast cancer cells modestly delayed primary tumor growth but dramatically suppressed lung metastasis in vivo. Furthermore, IL13Ralpha2 silencing was associated with enhanced IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and impaired migratory ability of metastatic breast cancer cells. Interestingly, genome-wide transcriptional analysis revealed that IL13Ralpha2 knockdown and IL-13 treatment cooperatively upregulated the metastasis suppressor tumor protein 63 (TP63) in a STAT6-dependent manner. These observations are consistent with increased metastasis-free survival of breast cancer patients with high levels of TP63 and STAT6 expression and suggest that the STAT6-TP63 pathway could be involved in impairing metastatic dissemination of breast cancer cells to the lungs. Conclusion Our findings indicate that IL13Ralpha2 could be used as a promising biomarker to predict patient outcome and provide a rationale for assessing the efficacy of anti-IL13Ralpha2 therapies in a subset of highly aggressive basal-like breast tumors as a strategy to prevent metastatic disease. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0607-y) contains supplementary material, which is available to authorized users.

Myc promoter-binding protein-1 (MBP-1) is a shorter protein variant of the glycolytic enzyme alpha-enolase. Although several lines of evidence indicate that MBP-1 acts as a tumor suppressor, the cellular mechanisms and signaling pathways underlying MBP-1 expression still remain largely elusive. To dissect these pathways, we used the SkBr3 breast cancer cell line and non-tumorigenic HEK293T cells ectopically overexpressing alpha-enolase/MBP-1. Here, we demonstrate that induced cell stresses promote MBP-1 expression through the AKT/PERK/eIF2α signaling axis. Our results contribute to shedding light on the molecular mechanisms underlying MBP-1 expression in non-tumorigenic and cancer cells.

Intact p73 function is shown to be an important determinant of cellular sensitivity to anticancer agents. Inhibition of p73 function by dominant-negative proteins or by mutant p53 abrogates apoptosis and cytotoxicity induced by these agents. A polymorphism encoding either arginine (72R) or proline (72P) at codon 72 of p53 influences inhibition of p73 by a range of p53 mutants identified in squamous cancers. Clinical response following cisplatin-based chemo-radiotherapy for advanced head and neck cancer is influenced by this polymorphism, cancers expressing 72R mutants having lower response rates than those expressing 72P mutants. Polymorphism in p53 may influence individual responsiveness to cancer therapy.

This study was undertaken to investigate the expression and predictive value for outcome of multidrug resistance-associated (MDR) proteins P-glycoprotein (Pgp), MRP1, BCRP, and LRP, in advanced testicular germ-cell tumours (TGCT). Paraffin-embedded sections from 56 previously untreated patients with metastatic TGCT were immunostained for Pgp, MRP1, BCRP, and LRP. All patients received platinum-based chemotherapy after orchidectomy. Immunostaining was related to clinicopathological parameters, response to chemotherapy, and outcome. Strong and intermediate expressions of the different MDR-related proteins were: 27 and 41% (Pgp), 54 and 37% (MRP1), 86 and 7% (BCRP), and 14 and 29% (LRP). P-glycoprotein and MRP1 associated, respectively, to low AFP (P=0.026) and high LDH levels (P=0.014), whereas LRP expression associated with high beta-hCG levels (P=0.003) and stage IV tumours (P=0.029). No correlation was found between Pgp, MRP1, and BCRP expression and response to chemotherapy and survival. In contrast, patients with LRP-positive tumours (strong or intermediate expression) had shorter progression-free (P=0.0006) and overall survival (P=0.0116) than LRP-negative patients, even after individual log-rank adjustments by statistically associated variables. Our data suggest that a positive LRP immunostaining at the time of diagnosis in metastatic TGCT is associated with an adverse clinical outcome.

Background The p53 protein family, comprising p53, p63 and p73, is primarily involved in preserving genome integrity and preventing tumor onset, and also affects a range of physiological processes. Signal-dependent modifications of its members and of other pathway components provide cells with a sophisticated code to transduce a variety of stress signaling into appropriate responses. TP53 mutations are highly frequent in cancer and lead to the expression of mutant p53 proteins that are endowed with oncogenic activities and sensitive to stress signaling. Scope of review p53 family proteins have unique structural and functional plasticity, and here we discuss the relevance of prolyl-isomerization to actively shape these features. Major conclusions The anti-proliferative functions of the p53 family are carefully activated upon severe stress and this involves the interaction with prolyl-isomerases. In particular, stress-induced stabilization of p53, activation of its transcriptional control over arrest- and cell death-related target genes and of its mitochondrial apoptotic function, as well as certain p63 and p73 functions, all require phosphorylation of specific S/T-P motifs and their subsequent isomerization by the prolyl-isomerase Pin1. While these functions of p53 counteract tumorigenesis, under some circumstances their activation by prolyl-isomerases may have negative repercussions (e.g. tissue damage induced by anticancer therapies and ischemia-reperfusion, neurodegeneration). Moreover, elevated Pin1 levels in tumor cells may transduce deregulated phosphorylation signaling into activation of mutant p53 oncogenic functions. General significance The complex repertoire of biological outcomes induced by p53 finds mechanistic explanations, at least in part, in the association between prolyl-isomerases and the p53 pathway. This article is part of a Special Issue entitled Proline-directed foldases: Cell signaling catalysts and drug targets.

Oral cancer (OC) is a common and aggressive cancer with poor survival rate. Recent research has investigated in detail the complex network of chemical, viral, and (epi)genetic factors associated with oral carcinogenesis. However, the causal factors that trigger the oral carcinogenesis process and the molecular mechanisms that underlie the tumor progression, and metastasis of OC have not been clarified. The present study analyses the hypothesis that immune reactions following viral infections may crossreact with tumor suppressor action proteins involved in OC. Using proteome databases and epitope catalogue resources together with data derived from an ample scientific-clinical literature, potential peptide crossreactions between HCV, HPV16, EBV, and CMV pathogens and human p53 and CSMD tumor suppressor proteins are detailed. The present study might contribute to understand the molecular basis of oral carcinogenesis and open new therapeutic research perspectives.

The CDC73 gene, encoding parafibromin, has been identified as a tumour suppressor gene both in hyperparathyroidism-jaw tumour (HPT-JT) syndrome and in sporadic parathyroid carcinoma. While the vast majority of CDC73 mutations affect the N-terminus or the central core of the encoded protein, as yet few mutations have been reported affecting the C-terminus. Here, we report a case (Caucasian female, 28 years) with an invasive ossifying fibroma of the left mandible and hyperparathyroidism (sCa = 16 mg/dl, PTH = 660 pg/mL) due to a parathyroid lesion of 20 mm, hystologically diagnosed as carcinoma. The whole CDC73 gene was screened for the presence of mutations by Sanger sequencing. Immunohistochemistry, in vitro functional assays, Western blotting, MTT assays and in-silico modelling were performed to assess the effect of the detected mutation. Sequence analysis of the CDC73 gene in the proband revealed the presence of a novel deletion affecting the C-terminus of the encoded protein (c.1379delT/p.L460Lfs*18). Clinical and genetic analyses of the available relatives led to the identification of three additional carriers, one of whom was also affected by a parathyroid lesion. Immunohistochemistry, Western blotting, MTT and in-silico modelling assays revealed that the deletion leads to down-regulation of the mutated protein, most likely through a proteasome-mediated pathway. We also found that the deletion may cause a conformational change in the C-terminus of the protein, possibly affecting its interaction with partner proteins. Finally, we found that the mutant protein enhances cellular growth. We report a novel mutation in the CDC73 gene that may underlie HPT-JT syndrome. This mutation appears to affect the C-terminal moiety of the encoded protein, which is thought to interact with other protein partners. The identification of these partners may be instrumental for our understanding of the CDC73-associated phenotype.

Reversible protein phosphorylation is a central regulatory mechanism of cell function. Deregulation of the balanced actions of protein kinases and phosphatases has been frequently associated with several pathological conditions, including cancer. Many studies have already addressed the role of protein kinases misregulation in cancer. However, much less is known about protein phosphatases influence. Phosphoprotein Phosphatase 1 (PPP1) is one of the major serine/threonine protein phosphatases who has three catalytic isoforms: PPP1CA, PPP1CB, and PPP1CC. Its function is achieved by binding to regulatory subunits, known as PPP1-interacting proteins (PIPs), which may prefer a catalytic isoform. Also, some inhibitors/enhancers may exhibit isoform specificity. Here we show that, prodigiosin (PG), a molecule with anticancer properties, promotes the formation of PPP1CA-AKT complex and not of PPP1CC-MAPK complex. Both, AKT and MAPK, are wellknown PIPs from two pathways that crosstalk and regulate melanoma cells survival. In addition, the analysis performed using surface plasmon resonance (SPR) technology indicates that PPP1 interacts with obatoclax (OBX), a drug that belongs to the same family of PG. Overall, these results suggest that PG might, at least in part, act through PPP1C/PIPs. Also, this study is pioneer in demonstrating PPP1 isoform-specific modulation by small molecules. published

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti–α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti–α-enolase/low anti-annexin AI IgG2 and patients with low anti–α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti–α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum.

Contains fulltext : 136932.pdf (Publisher’s version ) (Open Access) This study aimed to determine whether telomere length (TL) is a marker of cancer risk or genetic status amongst two cohorts of BRCA1 and BRCA2 mutation carriers and controls. The first group was a prospective set of 665 male BRCA1/2 mutation carriers and controls (mean age 53 years), all healthy at time of enrollment and blood donation, 21 of whom have developed prostate cancer whilst on study. The second group consisted of 283 female BRCA1/2 mutation carriers and controls (mean age 48 years), half of whom had been diagnosed with breast cancer prior to enrollment. TL was quantified by qPCR from DNA extracted from peripheral blood lymphocytes. Weighted and unweighted Cox regressions and linear regression analyses were used to assess whether TL was associated with BRCA1/2 mutation status or cancer risk. We found no evidence for association between developing cancer or being a BRCA1 or BRCA2 mutation carrier and telomere length. It is the first study investigating TL in a cohort of genetically predisposed males and although TL and BRCA status was previously studied in females our results don't support the previous finding of association between hereditary breast cancer and shorter TL.

The tumor suppressor p53 is a transcription factor that coordinates the cellular response to several kinds of stress. p53 inactivation is an important step in tumor progression. Oligomerization of p53 is critical for its posttranslational modification and its ability to regulate the transcription of target genes necessary to inhibit tumor growth. Here we report that the HECT E3 ubiquitin ligase HERC2 interacts with p53. This interaction involves the CPH domain of HERC2 (a conserved domain within Cul7, PARC, and HERC2 proteins) and the last 43 amino acid residues of p53. Through this interaction, HERC2 regulates p53 activity. RNA interference experiments showed how HERC2 depletion reduces the transcriptional activity of p53 without affecting its stability. This regulation of p53 activity by HERC2 is independent of proteasome or MDM2 activity. Under these conditions, up-regulation of cell growth and increased focus formation were observed, showing the functional relevance of the HERC2-p53 interaction. This interaction was maintained after DNA damage caused by the chemotherapeutic drug bleomycin. In these stressed cells, p53 phosphorylation was not impaired by HERC2 knockdown. Interestingly, p53 mutations that affect its tetramerization domain disrupted the HERC2-p53 interaction, suggesting a role for HERC2 in p53 oligomerization. This regulatory role was shown using cross-linking assays. Thus, the inhibition of p53 activity after HERC2 depletion can be attributed to a reduction in p53 oligomerization. Ectopic expression of HERC2 (residues 2292-2923) confirmed these observations. Together, these results identify HERC2 as a novel regulator of p53 signaling.