Muc4 Modulation of Ligand-Independent ErbB2 Signaling

The membrane mucin Muc4 is a heterodimer, bi-functional glycoprotein complex that is normally expressed in epithelial tissue. Functional studies on the extracellular mucin subunit of Muc4 have shown that it acts to promote anti-adhesion properties by sterically interfering with cell-cell and cell-matrix interactions and that the extent of this effect is directly associated with the number of tandem repeats on this subunit. Functional studies on the transmembrane subunit of Muc4 have shown that this subunit participates in intracellular signaling through interaction with the receptor tyrosine kinase ErbB2. This role of Muc4 was shown to be mediated by stabilizing the heregulin ligand-induced ErbB2-ErbB3 heterodimer through interference with the internalization process of these receptors, thus potentiating the PI3K, a survival-signaling pathway that is mediated by this heterodimer. However, Muc4 was also shown to potentiate ErbB2 phosphorylation in the absence of heregulin by an unknown mechanism. The aim of this work was to examine the role of Muc4 in intracellular signaling by evaluating the ligand-independent Muc4-ErbB2 interaction. Biochemical analyses of A375 human melanoma cells expressing Muc4 under different cell treatments, and probed with phospho-specific antibodies, were used to understand the mechanism. An antibody microarray screen was used to decipher the intracellular activated signaling pathways. The results of the mechanistic analysis indicated that Muc4 potentiates ErbB2 signaling significantly by interacting with ErbB2 and ErbB3 and by stabilizing the kinase active ErbB2 receptor, thus increasing its phosphorylation signal half-life and resulting in sustained ErbB2 signaling. The signaling pathway analysis suggests that through Muc4 direct interaction with ErbB2, signaling pathways that promote loss of cell polarity are activated. Loss of cell-cell adhesion is mediated by interference with the cadherin-catenin complex stability, and loss of cell-matrix adhesion is mediated by facilitating focal adhesion turnover. Together, these results suggest that Muc4 is a potent oncogenic factor, and further enhance our understanding of the role that Muc4 plays in ligand-independent intracellular signaling

LymphomiRs: microRNAs with regulatory roles in lymphomas

This review provides current knowledge on the role of microRNAs (miRNAs) in lymphoma with an emphasis on mature B-cell lymphoma. Although miRNAs were previously used to stratify lymphoma classification, prognosis, or treatment response, recent publications portray this class of small noncoding RNAs as critical players in the lymphomagenesis process. Although functional studies provide ample evidence for their role as lymphoma drivers or suppressors, genetic studies providing the underlying mechanisms for these phenotypes are still lacking. As more whole-genome sequencing of lymphoma cases become available, this gap may be soon filled. Current findings highlight the potential role of miRNAs molecules as important factors in lymphomagenesis

MiR-181a Is a Master Regulator of the Nuclear Factor-κB Signaling Pathway in Diffuse Large B Cell Lymphoma

Abstract Abstract 417 MicroRNAs (miRNAs) exhibit differential expression in cancer and can be used as prognostic biomarkers. MiR-181a expression is reported to be associated with survival and outcome in acute myeloid and chronic lymphocytic leukemia patients. We demonstrated that miR-181a levels are independently associated with improved survival of diffuse large B cell lymphoma (DLBCL) patients treated with R-CHOP (Rituximab, Cyclophosphamide, Adriamycin, Oncovin, Prednisolone). However, the mechanism underlying this observation and the function of miR-181a in DLBCL pathogenesis are unknown. MiR-181a was expressed at higher levels in centroblasts compared to naïve and memory B cells, and at significantly higher levels in GCB-like compared to ABC-like DLBCL cell lines (p=0.017). These observations suggested that miR-181a may differentially target critical signaling pathways in GCB and ABC DLBCL. NF-kB serves a critical role in ABC DLBCL survival. Utilizing 3 miRNA target prediction algorithms, multiple NF-κB signaling pathway transcripts harbored putative miR-181a binding sites. Consequently, we tested the effect of miR-181a on CARD11, IBKα, p105/p50, and C-Rel expression in DLBCL cell lines (HBL1, VAL). Compared with a scrambled miRNA control, miR-181a expression decreased protein and mRNA levels of these targets. To confirm the effect was direct, we fused the 3′-UTR sequences of CARD11, IBKα, p105 and C-Rel, each containing miR-181a putative binding sites, to a luciferase reporter gene. Co-transfecting miR-181a with the corresponding constructs, we demonstrated that all the constructs had significantly repressed luciferase activity compared with a non-targeting control. The effect was specific, since miR-181a did not affect luciferase activity of CARD11, IBKα, p105 and C-Rel reporter constructs with mutated binding sites. Using an NF-κB luciferase reporter assay, we next demonstrated that compared to a scrambled control, miR-181a significantly decreased NF-κB reporter activity in DLBCL cell lines (VAL, SUDHL6, OCILY7, OCILY19, HBL1, RCK8). MiR-181a also decreased NF-κB reporter activity induced by anti-IgM and TNFα stimulation. Concordantly, anti-miR-181a increased endogenous p105/p50 and C-Rel protein levels. Because ubiquitinated-IKKγ drives NF-κB signaling, we tested the effect of miR-181a in TNFα-stimulated 293T cells on ubiquitinated-IKKγ. MiR-181a decreased levels of ubiquitinated-IKKγ, corroborating the observed inhibitory effects on NF-κB signaling. We reasoned that NF-κB signaling repression should coincide with a decrease in endogenous transcription activity from NF-κB promoters. Indeed, miR-181a decreased mRNA expression levels of NF-κB target genes (BCL2, IRF4, IL-6, IKBa, FN1, PIM1, BLR1, CCL3, CFLAR, FCER2, TP53) as measured by qRT-PCR in miR-181a-transfected HBL1 cells. Because miR-181a directly targets p105/p50 and REL proteins, we postulated that this may be one of the main mechanisms of NF-κB signaling repression. Indeed, an electrophoresis mobility shift assay along with super-shifts analyses showed a decrease in the p105/p50 protein in HeLa nuclear extracts. To examine the biological significance of differential miR-181a expression between GCB- and ABC-like DLBCL and elucidate its potential role in DLBCL pathogenesis, we next assessed cell death (Annexin V, 7AAD) and cell proliferation (BrdU, 7AAD) in GCB (SUDHL4, OCILY7, OCILY19, VAL) and ABC (HBL1, OCILY10, RCK8, U2932) DLBCL cell lines transfected with GFP labeled precursor miR-181a. MiR-181a expression significantly increased cell death and apoptosis of ABC versus GCB DLBCL (p=0.006). This was associated with a more pronounced G1 phase growth arrest in the ABC DLBCL cells. Our studies demonstrate that miR-181a is a master regulator of canonical NF-kB signaling by regulating the expression of multiple components of this pathway, an effect that may underlie the distinct prognosis of DLBCL with different miR-181a expression levels. Furthermore, miR-181a down regulation may contribute to the pathogenesis of ABC DLBCL. Disclosures: No relevant conflicts of interest to declare

Mirna-181a expression Lead to Longer Animal Survival and Slower Tumor-Growth Rate in Diffuse Large B-Cell Lymphoma Xenograft Models

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is subdivided into the germinal center B-like (GCB) and activated B cell-like (ABC) subtypes by gene expression profiling, and these subtypes exhibit different clinical outcomes and signaling pathway deregulations. Compared to the GCB, the ABC-DLBCL subtype displays a more aggressive clinical course and shorter patient survival. Constitutive nuclear factor kappa-B (NF-kB) activity is often associated with the ABC-DLBCL subtype, however recent studies suggest that NF-kB signaling activation is also observed to a lower extent in the GCB-DLBCL subtype (Lina Odqvist et al. 2014). miRNAs have diagnostic and prognostic value in disease classification, and growing evidence implicates miRNAs in tumorigenesis, tumor maintenance, and dissemination through their ability to modulate the expression of critical genes and signaling networks. We previously demonstrated that miRNA-181a expression correlates with longer survival in patients treated with R-CHOP, independent of established clinical and molecular predictors. However, the molecular and cellular mechanisms underlying the association between miRNA-181a expression and improved prognosis in DLBCL patients are currently unknown. Herein we analyzed the role of miRNA-181a in DLBCL pathogenesis. Results:Quantitative RT-PCR analyses demonstrate higher endogenous miRNA-181a levels in centroblasts than in plasmablasts. Concordantly, endogenous miRNA-181a levels were significantly higher in GCB DLBCL cell lines and primary tumors compared with ABC DLBCL. These expression differences could not be attributed to distinct DNA methylation signatures in the miRNA-181a promoters (Chromosomes 1, 9) or regulatory elements as analyzed by Mass Array Sequenom Epityping. In search for putative miRNA-181a targets we identified 5 genes (CARD11, NFKB1A (IKBα), NFKB1 (p105/p50), RELA (p65), and REL (CREL)) within the NF-kB signaling pathway. Analyses of these targets show a decrease in the levels of these proteins and mRNAs in ABC and GCB DLBCL cell lines ectopically expressing miRNA-181a compared with scramble control plasmid. Luciferase reporter analyses encoding the respective wild type or mutated 3′UTR sequences demonstrate direct and specific targeting of these transcripts with the exception of RELA. Analysis of the net effect of miRNA-181a on NF-kB signaling using NF-kB luciferase reporter demonstrate significant decrease in NF-kB signaling. Concordantly, anti-miRNA-181a transfection led to increased NF-kB luciferase reporter activity. Moreover, western blot analyses of cytoplasmic and nuclear fractions showed a decrease in the levels of the transcription factors CREL and p50 in both cellular compartments, a decrease in the binding to DNA at NF-kB binding motifs, and a consequent decrease in NF-kB target gene transcription in the miRNA-181a expressing cells compared with scramble control. Together these studies point to miRNA-181a-mediated repression of NF-kB signaling in DLBCLs. Ectopic miRNA-181a expression led to a decrease in cell proliferation and an increase in cell death in both DLBCL subtypes, but this effect was more pronounced in the ABC DLBCL cell lines. The miRNA-181a-mediated increase in cell apoptosis could not be rescued by BCL2 co-transfection, an anti-apoptotic protein that was previously established as a direct miRNA-181a target. Analyses of miRNA-181a effects in NOD/SCID mice demonstrated that in vivo miRNA-181a induction in GCB and ABC human DLBCL xenografts led to decreased tumor growth and significantly longer animal survival. Notably, survival was prolonged in both GCB and ABC DLBCL bearing animals. Figure 1 Figure 1. Conclusions: miRNA-181a directly suppress the NF-kB signaling pathway and lead to increased tumor cell death in both DLBCL subtypes suggesting that NF-kB deregulation is present in both tumor subtypes. However, the lower miRNA-181a expression level in the ABC DLBCL subtype may contribute to the higher NF-kB signaling activity that is observed in this subtype. Furthermore, our study provides a plausible explanation for the association between high miRNA-181a expression and longer survival of DLBCL patients. Disclosures No relevant conflicts of interest to declare

MicroRNAs Are Independent Predictors of Outcome in Diffuse Large B-Cell Lymphoma Patients Treated with R-CHOP

PURPOSE: Diffuse large B-cell lymphoma (DLBCL) heterogeneity has prompted investigations for new biomarkers that can accurately predict survival. A previously reported 6-gene model combined with the international prognostic index (IPI) could predict patients’ outcome. However, even these predictors are not capable of unambiguously identifying outcome, suggesting that additional biomarkers might improve their predictive power. EXPERIMENTAL DESIGN: We studied expression of 11 microRNAs that had previously been reported to have variable expression in DLBCL tumors. We measured the expression of each microRNA by quantitative real-time polymerase-chain-reaction analyses in 176 samples from uniformly treated DLBCL patients and correlated the results to survival. RESULTS: In a univariate analysis, the expression of miR-18a correlated with overall survival (OS), whereas the expression of miR-181a and miR-222 correlated with progression-free survival (PFS). A multivariate Cox regression analysis including the IPI, the 6-gene model-derived Mortality Predictor Score and expression the of miR-18a, miR-181a, and miR-222, revealed that all variables were independent predictors of survival except the expression of miR-222 for OS and the expression of miR-18a for PFS. CONCLUSION: The expression of specific miRNAs may be useful for DLBCL survival prediction and their role in the pathogenesis of this disease should be examined further

miR-181a negatively regulates NF-κB signaling and affects activated B-cell–like diffuse large B-cell lymphoma pathogenesis

Distinct subgroups of diffuse large B-cell lymphoma (DLBCL) genetically resemble specific mature B-cell populations that are blocked at different stages of the immune response in germinal centers (GCs). The activated B-cell (ABC)-like subgroup resembles post-GC plasmablasts undergoing constitutive survival signaling, yet knowledge of the mechanisms that negatively regulate this oncogenic signaling remains incomplete. In this study, we report that microRNA (miR)-181a is a negative regulator of nuclear factor κ-light-chain enhancer of activated B-cells (NF-κB) signaling. miR-181a overexpression significantly decreases the expression and activity of key NF-κB signaling components. Moreover, miR-181a decreases DLBCL tumor cell proliferation and survival, and anti-miR-181a abrogates these effects. Remarkably, these effects are augmented in the NF-κB dependent ABC-like subgroup compared with the GC B-cell (GCB)-like DLBCL subgroup. Concordantly, in vivo analyses of miR-181a induction in xenografts results in slower tumor growth rate and prolonged survival in the ABC-like DLBCL xenografts compared with the GCB-like DLBCL. We link these outcomes to relatively lower endogenous miR-181a expression and to NF-κB signaling dependency in the ABC-like DLBCL subgroup. Our findings indicate that miR-181a inhibits NF-κB activity, and that manipulation of miR-181a expression in the ABC-like DLBCL genetic background may result in a significant change in the proliferation and survival phenotype of this malignancy

Active IKKβ promotes the stability of GLI1 oncogene in diffuse large B-cell lymphoma

GLI1 oncogene has been implicated in the pathobiology of several neoplasms including diffuse large B-cell lymphoma (DLBCL). However, mechanisms underlying GLI1-increased activity in DLBCL are poorly characterized. Herein, we demonstrate that IKKβ phosphorylates GLI1 in DLBCL. IKKβ activation increased GLI1 protein levels and transcriptional activity, whereas IKKβ silencing decreased GLI1 levels and transcriptional activity. Tumor necrosis factor-α (TNFα) mediated IKKβ activation–impaired GLI1 binding with the E3 ubiquitin ligase-ITCH, leading to decreased K48-linked ubiquitination/degradation of GLI1. We found 8 IKKβ-dependent phosphorylation sites that mediate GLI1 stability. Mutating or deleting these residues facilitated GLI1-ITCH interaction and decreased the protective effect of TNFα on GLI1 stability. IKKβ-GLI1 crosstalk is significant because combined inhibition of both molecules resulted in synergistic suppression of DLBCL viability in vivo and in vitro. By linking IKKβ-mediated nuclear factor-κB activity with GLI1, we identified a crosstalk between these 2 pathways that can inform the design of novel therapeutic strategies in DLBCL