Circ_0058106 promotes proliferation, metastasis and EMT process by regulating Wnt2b/ß-catenin/c-Myc pathway through miR-185-3p in hypopharyngeal squamous cell carcinoma.

Hypopharyngeal squamous cell carcinoma (HSCC) accounts 95% of hypopharyngeal cancer, which is characterized by high early metastasis rate and poor prognosis. It is reported that circular RNA is involved in the occurrence and development of cancer; however, the role of circRNA in hypopharyngeal cancer has little been investigated. We performed hypopharyngeal carcinoma circRNA microarray and qRT-PCR verification. The results showed circ_0058106 expression level was significantly upregulated in tumor tissues than in corresponding normal tissues. We found that circ_0058106 upregulation promoted proliferation, migration and invasion of HSCC cells, while knockdown of circ_0058106 inhibited proliferation, migration and invasion of HSCC cells both in vitro and in vivo. Bioinformatics predicted circ_0058106 may interact with miR-185-3p. We verified circ_0058106 directly bound miR-185-3p and downregulated miR-185-3p expression by using dual-luciferase reporter assay and qRT-PCR. Moreover, we proved circ_0058106 promoted HSCC cells tumorigenesis and EMT process by regulating Wnt2b/ß-catenin/c-Myc pathway via miR-185-3p. In conclusion, our findings firstly confirmed the carcinogenic effect of circ_0058106 in promoting HSCC cells tumorigenesis, metastasis, invasion and EMT process by regulating Wnt2b/ß-catenin/c-Myc pathway through sponging miR-185-3p, indicating that circ_0058106 may be a new therapeutic target and prognostic marker for HSCC.

Impact of oncogenic pathways on evasion of antitumour immune responses.

Immunotherapeutic interventions are showing effectiveness across a wide range of cancer types, but only a subset of patients shows clinical response to therapy. Responsiveness to checkpoint blockade immunotherapy is favoured by the presence of a local, CD8+ T cell-based immune response within the tumour microenvironment. As molecular analyses of tumours containing or lacking a productive CD8+ T cell infiltrate are being pursued, increasing evidence is indicating that activation of oncogenic pathways in tumour cells can impair induction or execution of a local antitumour immune response. This Review summarizes our current knowledge of the influence of oncogenic effects on evasion of antitumour immunity.

Inactivation of MYC reverses tumorigenesis.

The MYC proto-oncogene is an essential regulator of many normal biological programmes. MYC, when activated as an oncogene, has been implicated in the pathogenesis of most types of human cancers. MYC overexpression in normal cells is restrained from causing cancer through multiple genetically and epigenetically controlled checkpoint mechanisms, including proliferative arrest, apoptosis and cellular senescence. When pathologically activated in the correct epigenetic and genetic contexts, MYC bypasses these mechanisms and drives many of the 'hallmark' features of cancer, including uncontrolled tumour growth associated with DNA replication and transcription, cellular proliferation and growth, protein synthesis and altered cellular metabolism. MYC also dictates tumour cell fate by enforcing self-renewal and by abrogating cellular senescence and differentiation programmes. Moreover, MYC influences the tumour microenvironment, including activating angiogenesis and suppressing the host immune response. Provocatively, brief or even partial suppression of MYC back to its physiological levels of activation can lead to the restoration of intrinsic checkpoint mechanisms, resulting in acute and sustained tumour regression associated with tumour cells undergoing proliferative arrest, differentiation, senescence and apoptosis, as well as remodelling of the tumour microenvironment, recruitment of an immune response and shutdown of angiogenesis. Hence, tumours appear to be addicted to the MYC oncogene because of both tumour cell intrinsic and host-dependent mechanisms. MYC is important for the regulation of both the initiation and maintenance of tumorigenesis.

The proto-oncogene MYC is required for selection in the germinal center and cyclic reentry.

After antigenic challenge, B cells enter the dark zone (DZ) of germinal centers (GCs) to proliferate and hypermutate their immunoglobulin genes. Mutants with greater affinity for the antigen are positively selected in the light zone (LZ) to either differentiate into plasma and memory cells or reenter the DZ. The molecular circuits that govern positive selection in the GC are not known. We show here that the GC reaction required biphasic regulation of expression of the cell-cycle regulator c-Myc that involved its transient induction during early GC commitment, its repression by Bcl-6 in DZ B cells and its reinduction in B cells selected for reentry into the DZ. Inhibition of c-Myc in vivo led to GC collapse, which indicated an essential role for c-Myc in GCs. Our results have implications for the mechanism of GC selection and the role of c-Myc in lymphomagenesis.

Anti-DR5 mAb ameliorate adjuvant arthritis rats through inducing synovial cells apoptosis.

OBJECTIVE: Study the therapeutic effects and immunoregulatory mechanisms of anti-DR5 mAb on adjuvant arthritis (AA) rats. METHODS: AA rats induced by CFA, were treated with anti-DR5 mAb through mainline administration. Effect on the synovial membranes of the tissues was detected by H&E staining. Flow cytometry and MTT assay were used for detecting the induced apoptosis in an in vitro system and TUNEL assay was used for analysis in an in vivo system. The involvement of the apoptotic pathway was further proved by a caspase inhibition assay. RESULTS: Anti-DR5 mAb could induce synovial cell apoptosis in an in vitro system, which was related with the mRNA expression of DR5 on the cell surface. The mRNA expressions of c-myc and bcl-2 were decreased in synovial cells and those of p21, p53, and bax were increased. The protein expressions of caspase-8/3/9, RANKL, JNK2, and c-Jun were raised and that of bcl-2 was decreased. When the caspase inhibitor was added to the synovial cells treated with anti-DR5 mAb, it showed a dose-dependence inhibition effect, indicating that anti-DR5 mAb inducing apoptosis might be through the caspase pathway. CONCLUSION: This study shows that anti-DR5 mAb can ameliorate arthritic symptoms. The mechanisms of the treatment are related to the increase in synovial cell apoptosis by regulating the mRNA expression of DR5 and apoptosis-related genes, prolonging the duration of the cell cycle by modulation of the mRNA expression of cell cycle-related genes, and the protein expression of the molecules in the caspase pathway and RANKL, JNK2, and c-Jun.

Restricting activation-induced cytidine deaminase tumorigenic activity in B lymphocytes.

DNA breaks play an essential role in germinal centre B cells as intermediates to immunoglobulin class switching, a recombination process initiated by activation-induced cytidine deaminase (AID). Immunoglobulin gene hypermutation is likewise catalysed by AID but is believed to occur via single-strand DNA breaks. When improperly repaired, AID-mediated lesions can promote chromosomal translocations (CTs) that juxtapose the immunoglobulin loci to heterologous genomic sites, including oncogenes. Two of the most studied translocations are the t(8;14) and T(12;15), which deregulate cMyc in human Burkitt's lymphomas and mouse plasmacytomas, respectively. While a complete understanding of the aetiology of such translocations is lacking, recent studies using diverse mouse models have shed light on two important issues: (1) the extent to which non-specific or AID-mediated DNA lesions promote CTs, and (2) the safeguard mechanisms that B cells employ to prevent AID tumorigenic activity. Here we review these advances and discuss the usage of pristane-induced mouse plasmacytomas as a tool to investigate the origin of Igh-cMyc translocations and B-cell tumorigenesis.

Correlation of gamma-catenin expression with good prognosis in medulloblastomas.

OBJECT: Medulloblastoma is a malignant cerebellar tumor of childhood and is difficult to cure due to frequent cerebrospinal fluid dissemination. Amplification of the c-myc gene (4%) and messenger (mRNA) overexpression (50%) are known to be adverse prognostic indicators. Because mRNA overexpression cannot be explained by gene amplification alone, mechanisms other than gene amplification are postulated. Molecules on the Wnt signal pathway in primary tumors were examined. METHODS: Immunohistochemical and cytogenetic examinations of beta- and gamma-catenin, c-myc, N-myc, and cyclin D1 in 24 primary medulloblastomas were conducted, and their clinical relevance was evaluated. Cytoplasmic/membranous staining of beta- and gamma-catenin was detected in 19 (79%) and nine (37%) cases, respectively, and nuclear expression of cyclin D1 and c-myc was detected in six (25%) and 21 (83%) cases, respectively. The expression levels of gamma-catenin in Western blot analysis and immunohistochemistry were similar. By differential polymerase chain reaction, c-myc and N-myc were amplified separately in two large cell/anaplastic medulloblastomas. No cyclin D1 amplification, or beta- or gamma-catenin mutations were found. Kaplan-Meier analysis revealed no dissemination at diagnosis (Chang Grade M0) and gamma-catenin expression was correlated with good prognosis (p = 0.0002 and 0.003, respectively). Expression of gamma-catenin was also significant in the M0 group (p = 0.022). Expression of cyclin D1 showed a trend toward adverse outcome (p = 0.057) and all patients in whom cyclin D1 expression was found died of disease. CONCLUSIONS: Expression of gamma-catenin is of great prognostic value and its immunohistochemistry may be useful for further stratification of treatment. Cyclin D expression may have the potential to be an adverse prognostic indicator.

Differential c-Myc responsiveness to B cell receptor ligation in B cell-negative selection.

Responsiveness of c-Myc oncogene to B cell receptor ligation has been implicated in the induction of apoptosis in transformed and normal immature B cells. These studies provided compelling evidence to link the c-Myc oncogene with the process of negative selection in B-lymphocytes. However, in addition to apoptosis, B cell-negative selection has been shown to occur by secondary Ig gene rearrangements, a mechanism called receptor editing. In this study, we assessed whether differential c-Myc responsiveness to B cell receptor (BCR) ligation is associated with the mechanism of negative selection in immature B cells. Using an in vitro bone marrow culture system and an Ig-transgenic mouse model (3-83) we show here that c-Myc is expressed at low levels throughout B cell development and that c-Myc responsiveness to BCR ligation is developmentally regulated and increased with maturation. Furthermore, we found that the competence to mount c-Myc responsiveness upon BCR ligation is important for the induction of apoptosis and had no effect on the process of receptor editing. Therefore, this study suggests an important role of c-Myc in promoting and/or maintaining B cell development and that compartmentalization of B cell tolerance may also be developmentally regulated by differential c-Myc responsiveness.

v-Src induces Shc binding to tyrosine 63 in the cytoplasmic domain of the LDL receptor-related protein 1.

We recently observed that the LDL receptor-related protein 1 (LRP-1) is tyrosine phosphorylated in v-Src-transformed cells. Using a GST-fusion protein containing the cytoplasmic domain of LRP-1, we show that LRP-1 is a direct substrate for v-Src in vitro. To study LRP-1 phosphorylation in vivo, we constructed an LRP-1 minireceptor composed of the beta chain linked at the amino-terminus to a Myc epitope (Myc-LRPbeta). When expressed together with v-Src, Myc-LRPbeta becomes phosphorylated on tyrosine. Of the four tyrosine residues present in the cytoplasmic domain of LRP-1, only Tyr 63 is phosphorylated by v-Src in vivo or in vitro. Using fibroblasts deficient in Src, Yes and Fyn, we were able to show that there are multiple kinases present in the cell that can phosphorylate LRP-1. Tyrosine-phosphorylated LRP-1 associates with Shc, a PTB and SH2 domain containing signaling protein that is involved in the activation of Ras. Binding of the purified Shc PTB domain to Tyr 63 containing peptides shows that the interaction between LRP-1 and Shc is direct. We found that DAB, a PTB domain containing signaling protein that is involved in signaling by LDL receptor-related proteins in the nervous system, did not bind to full-length LRP-1. Our observations suggest that LRP-1 may be involved in normal and malignant signal transduction through a direct interaction with Shc adaptor proteins.

The role of the Ets2 transcription factor in the proliferation, maturation, and survival of mouse thymocytes.

In this study, we investigated the effects of Ets2 expression on the proliferation, maturation, and survival of thymocytes by establishing transgenic mice that specifically express Ets2 or a dominant negative form of Ets2, Deltaets2, in the thymus. We show that, in young animals, there are fewer T cells in Deltaets2 transgenic thymi and that the maturation of these T cells is affected at the CD4(-)CD8(-) double-negative to CD4(+)CD8(+) double-positive transition compared with wild-type littermate mice. Partial recovery in the number of thymocytes and full T cell maturation are restored with increasing age of Deltaets2 transgenic animals. However, thymocytes from adult Deltaets2 transgenic mice cultured ex vivo are more sensitive to cell death and to glucocorticoid-induced apoptosis than are T cells from control littermate mice. We also show that T cells from adult ets2 transgenic mice proliferate faster than their wild-type littermates. The proliferation and survival of these T cells are clearly affected upon apoptotic signals: glucocorticoid-induced apoptosis induces T cells from ets2 transgenic mice to continue to proliferate in vivo and to survive better ex vivo than T cells from control littermates. It has been shown that c-Myc expression is required for thymic proliferation and improves thymocyte survival of dexamethasone-treated animals. We show that the expression of c-Myc, an Ets2 target, is elevated in T cells freshly isolated from thymi of ets2 transgenic mice pretreated with dexamethasone. Together, these results show that Ets2 plays a role in the proliferation and survival of thymocytes, implicating a Myc-dependent pathway.

Diva/Boo is a negative regulator of cell death in human glioma cells.

Diva is a novel proapoptotic member of the Bcl-2 protein family which binds apoptosis activating factor-1 (APAF-1). Diva is identical with Boo which was identified as a novel antiapoptotic Bcl-2 family protein. Here, we report that Diva promotes the cell cycle exit of human glioma cells in response to serum deprivation and inhibits apoptosis of these cells induced by CD95 ligand or chemotherapeutic drugs. In glioma cells, Diva interferes with apoptotic signaling downstream of cytochrome c release, but upstream of caspase activation, consistent with an inhibitory effect on the mitochondrial amplification step involving the apoptosome and APAF-1.

Neuroblastic and Schwannian stromal cells of neuroblastoma are derived from a tumoral progenitor cell.

The coexistence of neuroblastic and Schwannian stromal (SS) cells in differentiating neuroblastoma (NB), and derivation of Schwannian-like cells from neuroblastic clones in vitro, were accepted previously as evidence of a common pluripotent tumor stem line. This paradigm was challenged when SS cells were suggested to be reactive in nature. The advent of microdissection techniques, PCR-based allelic analysis, and in situ fluorescent cytometry made possible the analysis of pure cell populations in fresh surgical specimens, allowing unequivocal determination of clonal origins of various cell subtypes. To overcome the complexity and heterogeneity of three-dimensional tissue structure, we used: (a) Laser-Capture Microdissection to obtain histologically homogeneous cell subtype populations for allelotype analysis at chromosomes 1p36, 11q23, 14q32, and 17q and study of MYCN copy number; (b) multiparametric analysis by Laser-Scanning Cytometry of morphology, DNA content, and immunophenotype of intact cells from touch imprints; and (c) bicolor fluorescence in situ hybridization on touch imprints from manually microdissected neuroblast and stroma-rich areas. Histologically distinct SS and neuroblastic cells isolated by Laser-Capture Microdissection had the same genetic composition in 27 of 28 NB analyzed by allelic imbalance and gene copy number. In all 20 cases studied by Laser-Scanning Cytometry, SS cells identified by morphology and S-100 immunostaining had identical DNA content and GD2-staining pattern as their neuroblastic counterparts. In 7 cases, fluorescence in situ hybridization demonstrated the same chromosomal makeup for SS and neuroblastic cells. These results provide unequivocal evidence that neuroblastic and SS cells in NB are derived from genetically identical neoplastic cells and support the classical paradigm that NB arises from tumoral cells capable of development along multiple lineages.

Antibody-induced shedding of CD44 from adherent cells is linked to the assembly of the cytoskeleton.

CD44 is a widely expressed integral membrane glycoprotein that serves as a specific adhesion receptor for the extracellular matrix glycosaminoglycan hyaluronan. CD44 participates in a variety of physiological and pathological processes through its role in cell adhesion. Under appropriate conditions, the ectodomain of CD44 is proteolytically removed from the cell surface. In this study we show that excessive CD44 shedding can be induced in mouse fibroblasts and monocytes upon exposure of these cells to a CD44-specific Ab immobilized on plastic, whereas treatment with phorbol ester induces significantly enhanced CD44 release from the monocytes only. CD44 shedding proceeds normally in fibroblasts and monocytes deficient in TNF-alpha converting enzyme (TACE), a sheddase involved in the processing of several substrates. Conversely, activation of the CD44 protease has no effect on the release of TNF-alpha from TACE-expressing cells, although the same metalloprotease inhibitor effectively blocks both TACE and the CD44 sheddase. Concomitant with anti-CD44 Ab- or phorbol ester-induced CD44 shedding, dramatic changes are observed in cell morphology and the structure of the actin cytoskeleton. Disruption of actin assembly with cytochalasin reduces CD44 shedding, but not the release of TNF-alpha. Moreover, pharmacological activation of Rho family GTPases Rac1 and Cdc42, which regulate actin filament assembly into distinct cytoskeletal structures, has a profound effect on CD44 release. We conclude that the CD44 sheddase and TACE are distinct enzymes, and that Ab- and phorbol ester-enhanced cleavage of CD44 is controlled in a cell type-dependent fashion by Rho GTPases through the cytoskeleton.

A cell surface-displayed anti-c-myc single-chain antibody: new perspectives for the genetic improvement of cellular tumor vaccines.

We have shown recently that cell surface-bound, single-chain Fv antibodies (scFv) are a powerful tool for the improvement of cellular tumor vaccines. To simplify this approach and to develop a general tool for the generation and improvement of cellular tumor vaccines, we chose an scFv against a peptide from the human proto-oncogene c-myc that could anchor any c-myc-tagged protein to the cell surface. The retroviral vector p50-Mx-neo (pMESV) was used to express scFv on the surface of the human melanoma line SkMel63. The cell-bound anti-c-myc scFv bound specifically to a soluble purified anti-CD28 scFv carrying a c-myc peptide-tag at its C terminus. Proof of principle was determined by incubating human peripheral blood lymphocytes with a mixture of (a) anti-c-myc-transfected SkMel63 cells binding the anti-CD28 scFv and (b) SkMel63 cells transfected with an anti-CD3 scFv. A clear synergistic effect on T-cell activation was observed that was comparable with that obtained in previous studies using SkMel63 cells transfected with the gene for the anti-CD28 scFv. As the cell surface-displayed anti-c-myc scFv can bind any c-myc-tagged protein of interest, this technique facilitates the genetic engineering of cellular vaccines for the therapy of virtually all human neoplasias.

The IL-2 receptor promotes lymphocyte proliferation and induction of the c-myc, bcl-2, and bcl-x genes through the trans-activation domain of Stat5.

Studies assessing the role of Stat5 in the IL-2 proliferative signal have produced contradictory, and thus inconclusive, results. One factor confounding many of these studies is the ability of IL-2R to deliver redundant mitogenic signals from different cytoplasmic tyrosines on the IL-2R beta-chain (IL-2Rbeta). Therefore, to assess the role of Stat5 in mitogenic signaling independent of any redundant signals, all cytoplasmic tyrosines were deleted from IL-2Rbeta except for Tyr510, the most potent Stat5-activating site. This deletion mutant retained the ability to induce Stat5 activation and proliferation in the T cell line CTLL-2 and the pro-B cell line BA/F3. A set of point mutations at or near Tyr510 that variably compromised Stat5 activation also compromised the proliferative signal and revealed a quantitative correlation between the magnitude of Stat5 activation and proliferation. Proliferative signaling by a receptor mutant with a weak Stat5 activating site could be rescued by overexpression of wt Stat5a or b. Additionally, the ability of this receptor mutant to induce c-myc, bcl-x, and bcl-2 was enhanced by overexpression of wt Stat5. By contrast, overexpression of a version of Stat5a lacking the C-terminal trans-activation domain inhibited the induction of these genes and cell proliferation. Thus, Stat5 is a critical component of the proliferative signal from Tyr510 of the IL-2R and regulates expression of both mitogenic and survival genes through its trans-activation domain.

Regulation of c-myc expression by IFN-gamma through Stat1-dependent and -independent pathways.

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50(cdc37) that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogated the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells.

Ongoing hypermutation in the Ig V(D)J gene segments and c-myc proto-oncogene of an AIDS lymphoma segregates with neoplastic B cells at different sites: implications for clonal evolution.

To investigate the role of somatic Ig hypermutation in the evolution of AIDS-associated B cell lymphomas, we analyzed the Ig V(D)J and c-myc genes expressed by neoplastic B cells in two extranodal sites, testis and orbit, and clonally related cells in the bone marrow. Testis and orbit B cells expressed differentially mutated but collinear V(H)DJ(H), V kappa J kappa and c-myc gene sequences. Shared mutations accounted for 10.2%, 8.4%, and 4.3% of the overall V(H)DJ(H), V kappa J kappa, and c-myc gene sequences. Tumor-site specific V(H)DJ(H), V kappa J kappa, and c-myc mutations were comparable in frequency, and a single point-mutation gave rise to an EcoRI site in the testis c-myc DNA. Both shared and tumor site-specific V(H)DJ(H), V kappa J kappa, and c-myc mutations displayed predominance of transitions over transversions. The "neoplastic" V(H)DJ(H) sequence was expressed by about 10(-5) cells in the bone marrow, and contained two of the three orbital, but none of the testicular V(H)DJ(H) mutations. The nature and distribution of the Ig V(D)J mutations found in the kappa chain suggested a selection by antigen in testis and orbit. Our data suggest that, in AIDS-associated B cell lymphomas, the Ig hypermutation machinery targets V(H)DJ(H), V kappa J kappa, and c-myc genes with comparable efficiency and modalities.