CBLB

Gene Summary

Gene:CBLB; Cbl proto-oncogene B
Aliases: Cbl-b, RNF56, Nbla00127
Location:3q13.11
Summary:This gene encodes an E3 ubiquitin-protein ligase which promotes proteosome-mediated protein degradation by transferring ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate. The encoded protein is involved in the regulation of immune response by limiting T-cell receptor, B-cell receptor, and high affinity immunoglobulin epsilon receptor activation. Studies in mouse suggest that this gene is involved in antifungal host defense and that its inhibition leads to increased fungal killing. Manipulation of this gene may be beneficial in implementing immunotherapies for a variety of conditions, including cancer, autoimmune diseases, allergies, and infections. [provided by RefSeq, Sep 2017]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:E3 ubiquitin-protein ligase CBL-B
Source:NCBIAccessed: 01 September, 2019

Ontology:

What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 01 September 2019 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 01 September, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CBLB (cancer-related)

Ishaque N, Abba ML, Hauser C, et al.
Whole genome sequencing puts forward hypotheses on metastasis evolution and therapy in colorectal cancer.
Nat Commun. 2018; 9(1):4782 [PubMed] Free Access to Full Article Related Publications
Incomplete understanding of the metastatic process hinders personalized therapy. Here we report the most comprehensive whole-genome study of colorectal metastases vs. matched primary tumors. 65% of somatic mutations originate from a common progenitor, with 15% being tumor- and 19% metastasis-specific, implicating a higher mutation rate in metastases. Tumor- and metastasis-specific mutations harbor elevated levels of BRCAness. We confirm multistage progression with new components ARHGEF7/ARHGEF33. Recurrently mutated non-coding elements include ncRNAs RP11-594N15.3, AC010091, SNHG14, 3' UTRs of FOXP2, DACH2, TRPM3, XKR4, ANO5, CBL, CBLB, the latter four potentially dual protagonists in metastasis and efferocytosis-/PD-L1 mediated immunosuppression. Actionable metastasis-specific lesions include FAT1, FGF1, BRCA2, KDR, and AKT2-, AKT3-, and PDGFRA-3' UTRs. Metastasis specific mutations are enriched in PI3K-Akt signaling, cell adhesion, ECM and hepatic stellate activation genes, suggesting genetic programs for site-specific colonization. Our results put forward hypotheses on tumor and metastasis evolution, and evidence for metastasis-specific events relevant for personalized therapy.

Sekiguchi N, Nomoto J, Nagata A, et al.
Gene Expression Profile Signature of Aggressive Waldenström Macroglobulinemia with Chromosome 6q Deletion.
Biomed Res Int. 2018; 2018:6728128 [PubMed] Free Access to Full Article Related Publications
Background: Waldenström macroglobulinemia (WM) is a rare, indolent B-cell lymphoma. Clinically, chromosome 6q deletion (6q del) including loss of the B lymphocyte-induced maturation protein 1 gene (BLIMP-1) is reported to be associated with poor prognosis. However, it remains unclear how the underlying biological mechanism contributes to the aggressiveness of WM with 6q del.
Methods: Here, we conducted oligonucleotide microarray analysis to clarify the differences in gene expression between WM with and without 6q del. Gene ontology (GO) analysis was performed to identify the main pathways underlying differences in gene expression. Eight bone marrow formalin-fixed paraffin-embedded samples of WM were processed for interphase fluorescence in situ hybridization analysis, and three were shown to have 6q del.
Results: GO analysis revealed significant terms including "lymphocyte activation" (corrected p value=6.68E-11), which included 31 probes. Moreover,
Conclusion: The present study suggested that the BCR signaling pathway and

Koba H, Kimura H, Nishikawa S, et al.
Next-generation sequencing analysis identifies genomic alterations in pathological morphologies: A case of pulmonary carcinosarcoma harboring EGFR mutations.
Lung Cancer. 2018; 122:146-150 [PubMed] Related Publications
OBJECTIVES: Pulmonary carcinosarcoma is a rare lung malignancy and little analysis has been performed to identify associated genomic alterations. We used next-generation sequencing (NGS) to analyze a pulmonary carcinosarcoma harboring an epidermal growth factor receptor (EGFR) mutation.
MATERIALS AND METHODS: The lung carcinosarcoma used for this study contained components of adenocarcinoma and chondrosarcoma and originated from a 73-year-old female. Both components carried deletion mutations in exon 19 of EGFR and both had equally strong EGFR protein expression. This study analyzed the biological and genetic characteristics of both components, using NGS and immunohistochemical (IHC) staining.
RESULTS AND CONCLUSION: IHC staining revealed that both total EGFR and deletion mutation specific EGFR proteins were equally expressed in both components. Intriguingly, identification of genomic alterations with NGS found five identical alterations in four genes (EGFR, CBLB, TP53, and MEN1) that were shared by the two components, and that each component had a large number of individual alterations. Additionally, we focused on an alpha-thalassemia/mental retardation syndrome X-linked (ATRX) mutation which was only present in the sarcoma component. ATRX protein expression was also only detected in the sarcoma component. This is the first report of the exhaustive genomic alterations in a pulmonary carcinosarcoma harboring an EGFR mutation. The results show that our case had the same EGFR status in both components. The EGFR mutation is the driver mutation in both components. In our case, we found that TP53 may be a common alteration and ATRX may be a specific alteration in the sarcoma component.

Li C, Dong Q, Che X, et al.
MicroRNA-29b-2-5p inhibits cell proliferation by directly targeting Cbl-b in pancreatic ductal adenocarcinoma.
BMC Cancer. 2018; 18(1):681 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: MicroRNAs can be used in the prognosis of malignancies; however, their regulatory mechanisms are unknown, especially in pancreatic ductal adenocarcinoma (PDAC).
METHODS: In 120 PDAC specimens, miRNA levels were assessed by quantitative real time polymerase chain reaction (qRT-PCR). Then, the role of miR-29b-2-5p in cell proliferation was evaluated both in vitro (Trypan blue staining and cell cycle analysis in the two PDAC cell lines SW1990 and Capan-2) and in vivo using a xenograft mouse model. Next, bioinformatics methods, a luciferase reporter assay, Western blot, and immunohistochemistry (IHC) were applied to assess the biological effects of Cbl-b inhibition by miR-29b-2-5p. Moreover, the relationship between Cbl-b and p53 was evaluated by immunoprecipitation (IP), Western blot, and immunofluorescence.
RESULTS: From the 120 PDAC patients who underwent surgical resection, ten patients with longest survival and ten with shortest survival were selected. We found that high miR-29b-2-5p expression was associated with good prognosis (p = 0.02). The validation cohort confirmed miR-29b-2-5p as an independent prognostic factor in PDAC (n = 100, 95% CI = 0.305-0.756, p = 0.002). Furthermore, miR-29b-2-5p inhibited cell proliferation, induced cell cycle arrest, and promoted apoptosis both in vivo and in vitro. Interestingly, miR-29b-2-5p directly bound the Cbl-b gene, down-regulating its expression and reducing Cbl-b-mediated degradation of p53. Meanwhile, miR-29b-2-5p expression was negatively correlated with Cbl-b in PDAC tissues (r = - 0.33, p = 0.001).
CONCLUSIONS: Taken together, these findings indicated that miR-29b-2-5p improves prognosis in PDAC by targeting Cbl-b to promote p53 expression, and would constitute an important prognostic factor in PDAC.

Li P, Liu H, Zhang Z, et al.
Expression and Comparison of Cbl-b in Lung Squamous Cell Carcinoma and Adenocarcinoma.
Med Sci Monit. 2018; 24:623-635 [PubMed] Free Access to Full Article Related Publications
BACKGROUND Non-small cell lung carcinoma (NSCLC) mainly includes lung squamous cell carcinoma and adenocarcinoma. This study aimed to investigate the difference between the expression of Cbl-b in lung squamous cell carcinoma and adenocarcinoma. MATERIAL AND METHODS The clinical features and survival data of NSCLC patients and Cbl-b mRNA (FPKM) were obtained from the TCGA database. Then, lung squamous cell carcinoma and adenocarcinoma cell lines were transfected with lentivirus-mediated RNA interference vector to knockdown the expression of Cbl-b. Next, a Transwell assay was performed to study the effect of Cbl-b shRNA on migration and invasion of lung squamous cell carcinoma and adenocarcinoma cells. Finally, Western blot analysis was performed to measure the expressions of PI3K, p-PI3K, AKT, p-AKT, ERK1/2, p-ERK1/2, GSK3β, p-GSK3β, mTOR, and p-mTOR protein in lung adenocarcinoma and squamous cell carcinoma cells. RESULTS The correlation of Cbl-b expression and OS was different between NSCLC adenocarcinoma and squamous carcinoma. After transfection, the expression of Cbl-b was inhibited in A549, H1975, and SW900 cells. Cbl-b shRNA promoted the migration and invasion of lung adenocarcinoma A549 and H1975 cells, but it inhibited the invasion of lung squamous cell carcinoma SW900 cells. In addition, Cbl-b regulated the expression of PI3K and ERK1/2-GSK3β pathway proteins in A549 and SW900 cells. CONCLUSIONS The OS of Cbl-b mRNA low expression in lung adenocarcinoma and squamous cell carcinoma was different. The difference in signal pathways may be one of the reasons for the difference in the correlation between Cbl-b expression and the survival rate of these 2 pathological types of lung cancer.

Jack J, Small GW, Brown CC, et al.
Gene expression and linkage analysis implicate CBLB as a mediator of rituximab resistance.
Pharmacogenomics J. 2018; 18(3):467-473 [PubMed] Free Access to Full Article Related Publications
Elucidating resistance mechanisms for therapeutic monoclonal antibodies (MAbs) is challenging, because they are difficult to study in non-human models. We therefore developed a strategy to genetically map in vitro drug sensitivity, identifying genes that alter responsiveness to rituximab, a therapeutic anti-CD20 MAb that provides significant benefit to patients with B-cell malignancies. We discovered novel loci with genome-wide mapping analyses and functionally validated one of these genes, CBLB, which causes rituximab resistance when knocked down in lymphoma cells. This study demonstrates the utility of genome-wide mapping to discover novel biological mechanisms of potential clinical advantage.

Robles-Valero J, Lorenzo-Martín LF, Menacho-Márquez M, et al.
A Paradoxical Tumor-Suppressor Role for the Rac1 Exchange Factor Vav1 in T Cell Acute Lymphoblastic Leukemia.
Cancer Cell. 2017; 32(5):608-623.e9 [PubMed] Free Access to Full Article Related Publications
Rho guanine exchange factors (GEFs), the enzymes that stimulate Rho GTPases, are deemed as potential therapeutic targets owing to their protumorigenic functions. However, the understanding of the spectrum of their pathobiological roles in tumors is still very limited. We report here that the GEF Vav1 unexpectedly possesses tumor-suppressor functions in immature T cells. This function entails the noncatalytic nucleation of complexes between the ubiquitin ligase Cbl-b and the intracellular domain of Notch1 (ICN1) that favors ICN1 ubiquitinylation and degradation. Ablation of Vav1 promotes ICN1 signaling and the development of T cell acute lymphoblastic leukemia (T-ALL). The downregulation of Vav1 is essential for the pathogenesis of human T-ALL of the TLX

Märklin M, Heitmann JS, Fuchs AR, et al.
NFAT2 is a critical regulator of the anergic phenotype in chronic lymphocytic leukaemia.
Nat Commun. 2017; 8(1):755 [PubMed] Free Access to Full Article Related Publications
Chronic lymphocytic leukaemia (CLL) is a clonal disorder of mature B cells. Most patients are characterised by an indolent disease course and an anergic phenotype of their leukaemia cells, which refers to a state of unresponsiveness to B cell receptor stimulation. Up to 10% of CLL patients transform from an indolent subtype to an aggressive form of B cell lymphoma over time (Richter´s syndrome) and show a significantly worse treatment outcome. Here we show that B cell-specific ablation of Nfat2 leads to the loss of the anergic phenotype culminating in a significantly compromised life expectancy and transformation to aggressive disease. We further define a gene expression signature of anergic CLL cells consisting of several NFAT2-dependent genes including Cbl-b, Grail, Egr2 and Lck. In summary, this study identifies NFAT2 as a crucial regulator of the anergic phenotype in CLL.NFAT2 is a transcription factor that has been linked with chronic lymphocytic leukaemia (CLL), but its functions in CLL manifestation are still unclear. Here the authors show, by analysing mouse CLL models and characterising biopsies from CLL patients, that NFAT2 is an important regulator for the anergic phenotype of CLL.

Lv K, Jiang J, Donaghy R, et al.
CBL family E3 ubiquitin ligases control JAK2 ubiquitination and stability in hematopoietic stem cells and myeloid malignancies.
Genes Dev. 2017; 31(10):1007-1023 [PubMed] Free Access to Full Article Related Publications
Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of

Xu L, Zhang Y, Qu X, et al.
E3 Ubiquitin Ligase Cbl-b Prevents Tumor Metastasis by Maintaining the Epithelial Phenotype in Multiple Drug-Resistant Gastric and Breast Cancer Cells.
Neoplasia. 2017; 19(4):374-382 [PubMed] Free Access to Full Article Related Publications
Multiple drug resistance (MDR) and metastasis are two major factors that contribute to the failure of cancer treatment. However, the relationship between MDR and metastasis has not been characterized. Additionally, the role of the E3 ubiquitin ligase Cbl-b in metastasis of MDR gastric and breast cancer is not well known. In the present study, we found that MDR gastric and breast cancer cells possess a typical mesenchymal phenotype and enhanced cell migration capacity. Additionally, Cbl-b is poorly expressed in MDR gastric and breast cancer cells. In MDR gastric adenocarcinoma tissues, gastric cancer patients with low Cbl-b expression were more likely to have tumor invasion (P=.016) and lymph node metastasis (P=.007). Moreover, overexpression of Cbl-b reduced cell migration in MDR cell cultures both in vitro and in vivo. Cbl-b overexpression also prevented EMT by inducing ubiquitination and degradation of EGFR, leading to inhibition of the EGFR-ERK/Akt-miR-200c-ZEB1 axis. However, further overexpression of EGFR on a background of Cbl-b overexpression restored both the mesenchymal phenotype and cell migration capacity of MDR gastric and breast cancer cells. These results suggest that Cbl-b is an important factor for maintenance of the epithelial phenotype and inhibition of cell migration in MDR gastric and breast cancer cells.

Mukhopadhyay C, Triplett A, Bargar T, et al.
Casitas B-cell lymphoma (Cbl) proteins protect mammary epithelial cells from proteotoxicity of active c-Src accumulation.
Proc Natl Acad Sci U S A. 2016; 113(51):E8228-E8237 [PubMed] Free Access to Full Article Related Publications
Casitas B-cell lymphoma (Cbl) family ubiquitin ligases negatively regulate tyrosine kinase-dependent signal transduction by promoting degradation of active kinases. We and others previously reported that loss of Cbl functions caused hyperproliferation in lymphoid and hematopoietic systems. Unexpectedly, Cbl deletion in Cbl-b-null, Cbl-c-null primary mouse mammary epithelial cells (MECs) (Cbl triple-deficiency) induced rapid cell death despite enhanced MAP kinase and AKT activation. Acute Cbl triple-deficiency elicited distinct transcriptional and biochemical responses with partial overlap with previously described cellular reactions to unfolded proteins and oxidative stress. Although the levels of reactive oxygen species were comparable, detergent-insoluble protein aggregates containing phosphorylated c-Src accumulated in Cbl triple-deficient MECs. Treatment with a broad-spectrum kinase inhibitor dasatinib blocked protein aggregate accumulation and restored in vitro organoid formation. This effect is most likely mediated through c-Src because Cbl triple-deficient MECs were able to form organoids upon shRNA-mediated c-Src knockdown. Taking these data together, the present study demonstrates that Cbl family proteins are required to protect MECs from proteotoxic stress-induced cell death by promoting turnover of active c-Src.

Dong Q, Li C, Che X, et al.
MicroRNA-891b is an independent prognostic factor of pancreatic cancer by targeting Cbl-b to suppress the growth of pancreatic cancer cells.
Oncotarget. 2016; 7(50):82338-82353 [PubMed] Free Access to Full Article Related Publications
Growing evidence has revealed that microRNAs could regulate the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells and predict the prognosis of PDAC. Here the comparative microRNA expression profiles of the good and poor prognosis groups were performed by microRNA microarray. MicroRNA-891b (miR-891b) was screened and validated to be a prognostic predictor of PDAC in the initial group and further evaluated to be an independent predictor for the overall survival of resectable PDACs in an independent cohort. By a series of cellular and animal experiments, as well as clinical specimen analyses, miR-891b was confirmed to target the Cbl-b gene, promot the expression of tumor suppressor p21 protein and inhibit the proliferation of PDAC cells. The results provide a theoretical basis for the study of miR-891b as an independent prognostic predictor of PDAC and the role of miR-891b/Cbl-b pathway in this prediction, as well as the identification of new targets for PDAC.

Prasad A, Rabionet R, Espinet B, et al.
Identification of Gene Mutations and Fusion Genes in Patients with Sézary Syndrome.
J Invest Dermatol. 2016; 136(7):1490-1499 [PubMed] Related Publications
Sézary syndrome is a leukemic form of cutaneous T-cell lymphoma with an aggressive clinical course. The genetic etiology of the disease is poorly understood, with chromosomal abnormalities and mutations in some genes being involved in the disease. The goal of our study was to understand the genetic basis of the disease by looking for driver gene mutations and fusion genes in 15 erythrodermic patients with circulating Sézary cells, 14 of them fulfilling the diagnostic criteria of Sézary syndrome. We have discovered genes that could be involved in the pathogenesis of Sézary syndrome. Some of the genes that are affected by somatic point mutations include ITPR1, ITPR2, DSC1, RIPK2, IL6, and RAG2, with some of them mutated in more than one patient. We observed several somatic copy number variations shared between patients, including deletions and duplications of large segments of chromosome 17. Genes with potential function in the T-cell receptor signaling pathway and tumorigenesis were disrupted in Sézary syndrome patients, for example, CBLB, RASA2, BCL7C, RAMP3, TBRG4, and DAD1. Furthermore, we discovered several fusion events of interest involving RASA2, NFKB2, BCR, FASN, ZEB1, TYK2, and SGMS1. Our work has implications for the development of potential therapeutic approaches for this aggressive disease.

Hinrichs CS
Molecular Pathways: Breaking the Epithelial Cancer Barrier for Chimeric Antigen Receptor and T-cell Receptor Gene Therapy.
Clin Cancer Res. 2016; 22(7):1559-64 [PubMed] Free Access to Full Article Related Publications
Adoptive transfer of T cells genetically engineered to express a tumor-targeting chimeric antigen receptor (CAR) or T-cell receptor (TCR) can mediate cancer regression in some patients. CARs are synthetic single-chain proteins that use antibody domains to target cell surface antigens. TCRs are natural heterodimeric proteins that can target intracellular antigens through recognition of peptides bound to human leukocyte antigens. CARs have shown promise in B-cell malignancies and TCRs in melanoma, but neither approach has achieved clear success in an epithelial cancer. Treatment of epithelial cancers may be particularly challenging because of a paucity of target antigens expressed by carcinomas and not by important healthy tissues. In addition, epithelial cancers may be protected by inhibitory ligands and soluble factors in the tumor microenvironment. One strategy to overcome these negative regulators is to modulate expression of T-cell genes to enhance intrinsic T-cell function. Programmable nucleases, which can suppress inhibitory genes, and inducible gene expression systems, which can enhance stimulatory genes, are entering clinical testing. Other work is delineating whether control of genes for immune checkpoint receptors (e.g.,PDCD1, CTLA4) and cytokine and TCR signaling regulators (e.g.,CBLB, CISH, IL12, IL15) can increase the antitumor activity of therapeutic T cells.

Terashima M, Togashi Y, Sato K, et al.
Functional Analyses of Mutations in Receptor Tyrosine Kinase Genes in Non-Small Cell Lung Cancer: Double-Edged Sword of DDR2.
Clin Cancer Res. 2016; 22(14):3663-71 [PubMed] Related Publications
PURPOSE: This study investigated whether mutations of receptor tyrosine kinase (RTK) genes detected using next-generation sequencing (NGS) are suitable therapeutic targets.
EXPERIMENTAL DESIGN: Fifty surgically resected non-small cell lung cancer (NSCLC) samples were target resequenced using NGS. We then investigated the functions of the identified RTK gene mutations, including their oncogenic potential, in vitro
RESULTS: Mutations in RTK genes were found in 20 samples (EGFR, 15; ERBB4, 1; ALK, 1; DDR2, 2; FGFR1, 1), mutations in MAPK pathway genes were found in nine samples (KRAS, 7; NRAS, 1; BRAF, 2), and mutations in PI3K pathway genes were found in three samples (PIK3CA, 1; PTEN, 3). Among the mutations in RTKs, the functions of four mutations were unclear (ERBB4 D245G; DDR2 H246R and E655K; FGFR1 A263V). These mutations did not exhibit any transformational activities. Neither the phosphorylation nor the protein expressions of RTKs were changed by the DDR2 H246R, ERBB4 D245G, and FGFR1 A263V mutations, although the expression level of the DDR2 protein harboring the E655K mutation was particularly low. Collagen stimulation decreased cellular proliferation through p38 activation in the DDR2 wild-type-overexpressed cell lines, whereas the growth-suppressive effect was weakened in DDR2 E655K-overexpressed cell lines. Furthermore, the DDR2 E655K protein strongly bound to ubiquitin ligase E3 (Cbl-b), and the mutant protein expression was increased after treatment with a proteasome inhibitor.
CONCLUSIONS: Our experimental findings suggest that RTK mutations are not always suitable as therapeutic targets. The DDR2 E655K mutation can play a role in cancer progression by reducing the growth-inhibitory effect of collagen. Clin Cancer Res; 22(14); 3663-71. ©2016 AACR.

Vennin C, Spruyt N, Dahmani F, et al.
H19 non coding RNA-derived miR-675 enhances tumorigenesis and metastasis of breast cancer cells by downregulating c-Cbl and Cbl-b.
Oncotarget. 2015; 6(30):29209-23 [PubMed] Free Access to Full Article Related Publications
H19 is a long non-coding RNA precursor of miR-675 microRNA. H19 is increasingly described to play key roles in the progression and metastasis of cancers from different tissue origins. We have previously shown that the H19 gene is activated by growth factors and increases breast cancer cell invasion. In this study, we established H19/miR-675 ectopic expression models of MDA-MB-231 breast cancer cells to further investigate the underlying mechanisms of H19 oncogenic action. We showed that overexpression of H19/miR-675 enhanced the aggressive phenotype of breast cancer cells including increased cell proliferation and migration in vitro, and increased tumor growth and metastasis in vivo. Moreover, we identified ubiquitin ligase E3 family (c-Cbl and Cbl-b) as direct targets of miR-675 in breast cancer cells. Using a luciferase assay, we demonstrated that H19, through its microRNA, decreased both c-Cbl and Cbl-b expression in all breast cancer cell lines tested. Thus, by directly binding c-Cbl and Cbl-b mRNA, miR-675 increased the stability and the activation of EGFR and c-Met, leading to sustained activation of Akt and Erk as well as enhanced cell proliferation and migration. Our data describe a novel mechanism of protumoral action of H19 in breast cancer.

Bunaciu RP, Jensen HA, MacDonald RJ, et al.
6-Formylindolo(3,2-b)Carbazole (FICZ) Modulates the Signalsome Responsible for RA-Induced Differentiation of HL-60 Myeloblastic Leukemia Cells.
PLoS One. 2015; 10(8):e0135668 [PubMed] Free Access to Full Article Related Publications
6-Formylindolo(3,2-b)carbazole (FICZ) is a photoproduct of tryptophan and an endogenous high affinity ligand for aryl hydrocarbon receptor (AhR). It was previously reported that, in patient-derived HL-60 myeloblastic leukemia cells, retinoic acid (RA)-induced differentiation is driven by a signalsome containing c-Cbl and AhR. FICZ enhances RA-induced differentiation, assessed by expression of the membrane differentiation markers CD38 and CD11b, cell cycle arrest and the functional differentiation marker, inducible oxidative metabolism. Moreover, FICZ augments the expression of a number of the members of the RA-induced signalsome, such as c-Cbl, Vav1, Slp76, PI3K, and the Src family kinases Fgr and Lyn. Pursuing the molecular signaling responsible for RA-induced differentiation, we characterized, using FRET and clustering analysis, associations of key molecules thought to drive differentiation. Here we report that, assayed by FRET, AhR interacts with c-Cbl upon FICZ plus RA-induced differentiation, whereas AhR constitutively interacts with Cbl-b. Moreover, correlation analysis based on the flow cytometric assessment of differentiation markers and western blot detection of signaling factors reveal that Cbl-b, p-p38α and pT390-GSK3β, are not correlated with other known RA-induced signaling components or with a phenotypic outcome. We note that FICZ plus RA elicited signaling responses that were not typical of RA alone, but may represent alternative differentiation-driving pathways. In clusters of signaling molecules seminal to cell differentiation, FICZ co-administered with RA augments type and intensity of the dynamic changes induced by RA. Our data suggest relevance for FICZ in differentiation-induction therapy. The mechanism of action includes modulation of a SFK and MAPK centered signalsome and c-Cbl-AhR association.

Cao Y, Qu J, Li C, et al.
Celecoxib sensitizes gastric cancer to rapamycin via inhibition of the Cbl-b-regulated PI3K/Akt pathway.
Tumour Biol. 2015; 36(7):5607-15 [PubMed] Related Publications
Mammalian target of rapamycin (mTOR) has emerged as a new potential therapeutic target for gastric cancer. However, a phase III clinical trial found that monotherapy with the mTOR inhibitor everolimus did not significantly improve the overall survival of patients with advanced gastric cancer. This has led to the exploration of more effective combinatorial regimens to enhance the effectiveness of mTOR inhibitors. Here, we demonstrate that Akt phosphorylation is increased in the rapamycin-resistant gastric cancer cell lines MGC803 and SGC7901. We further show that combined treatment with celecoxib and rapamycin results in an additive inhibitory effect on the growth of gastric cancer cells through suppression of rapamycin-induced Akt activation. Moreover, celecoxib upregulated the expression of the ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b). Knockdown of Cbl-b significantly attenuated celecoxib-mediated inhibition of Akt phosphorylation and impaired the additive anticancer effect of celecoxib and rapamycin. Our results suggest that celecoxib-mediated upregulation of Cbl-b is responsible, at least in part, for the additive antitumor effect of celecoxib and rapamycin via inhibition of rapamycin-induced Akt activation.

Kwak SY, Kim BY, Ahn HJ, et al.
Ionizing radiation-inducible miR-30e promotes glioma cell invasion through EGFR stabilization by directly targeting CBL-B.
FEBS J. 2015; 282(8):1512-25 [PubMed] Related Publications
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at the transcriptional and post-transcriptional levels. Here we show that miR-30e, which was previously identified as an ionizing radiation-inducible miRNA, enhances cellular invasion by promoting secretion of the matrix metalloproteinase MMP-2. The enhancement of cellular invasion by miR-30e involved up-regulation of the epidermal growth factor receptor (EGFR) and subsequent activation of its downstream signaling mediators, AKT and extracellular signal-regulated kinase. EGFR up-regulation by miR-30e occurred due to stabilization of the EGFR protein. The E3 ubiquitin ligase casitas B-lineage lymphoma B (CBL-B) was down-regulated by miR-30e, and this led to increased EGFR abundance. A 3' UTR reporter assay confirmed that CBL-B is a direct target of miR-30e. Knocking down CBL-B expression phenocopied the effects of miR-30e, whereas ectopic expression of CBL-B suppressed miR-30e-induced EGFR up-regulation and invasion. Collectively, our results suggest that targeting miR-30e may limit the invasiveness induced during glioma radiotherapy.

Huang C, Sheng Y, Jia J, Chen L
Identification of melanoma biomarkers based on network modules by integrating the human signaling network with microarrays.
J Cancer Res Ther. 2014; 10 Suppl:C114-24 [PubMed] Related Publications
BACKGROUND: Melanoma is a leading cause of cancer death. Thus, accurate prognostic biomarkers that will assist rational treatment planning need to be identified.
METHODS: Microarray analysis of melanoma and normal tissue samples was performed to identify differentially expressed modules (DEMs) from the signaling network and ultimately detect molecular markers to support histological examination. Network motifs were extracted from the human signaling network. Then, significant expression-correlation differential modules were identified by comparing the network module expression-correlation differential scores under normal and disease conditions using the gene expression datasets. Finally, we obtained DEMs by the Wilcoxon rank test and considered the average gene expression level in these modules as the classification features for diagnosing melanoma.
RESULTS: In total, 99 functional DEMs were identified from the signaling network and gene expression profiles. The area under the curve scores for cancer module genes, melanoma module genes, and whole network modules are 92.4%, 90.44%, and 88.45%, respectively. The classification efficiency rates for nonmodule features are 71.04% and 79.38%, which correspond to the features of cancer genes and melanoma cancer genes, respectively. Finally, we acquired six significant molecular biomarkers, namely, module 10 (CALM3, Ca 2+ , PKC, PDGFRA, phospholipase-g, PIB5PA, and phosphatidylinositol-3-kinase), module 14 (SRC, Src homology 2 domain-containing [SHC], SAM68, GIT1, transcription factor-4, CBLB, GRB2, VAV2, LCK, YES, PTCH2, downstream of tyrosine kinase [DOK], and KIT), module 16 (ELK3, p85beta, SHC, ZFYVE9, TGFBR1, TGFBR2, CITED1, SH3KBP1, HCK, DOK, and KIT), module 45 (RB, CCND3, CCNA2, CDK4, and CDK6), module 75 (PCNA, CDK4, and CCND1), and module 114 (PSD93, NMDAR, and FYN).
CONCLUSION: We explored the gene expression profile and signaling network in a global view and identified DEMs that can be used as diagnostic or prognostic markers for melanoma.

Hummel R, Sie C, Watson DI, et al.
MicroRNA signatures in chemotherapy resistant esophageal cancer cell lines.
World J Gastroenterol. 2014; 20(40):14904-12 [PubMed] Free Access to Full Article Related Publications
AIM: To investigate expression of microRNA (miRNA) and potential targets in chemotherapy resistant esophageal cancer cell lines.
METHODS: An in-vitro model of acquired chemotherapy resistance in esophageal adeno- (EAC) and squamous cell carcinoma (ESCC) cells was used, and microRNA expression profiles for cisplatin or 5-fluorouracil (5-FU) resistant variants vs chemotherapy sensitive controls were compared using microarray and quantitative real-time polymerase chain reaction (PCR). The expression of chemotherapy-relevant genes potentially targeted by the dysregulated microRNAs in the chemotherapy resistant variants was also evaluated.
RESULTS: Chemotherapy resistant sublines were found to have specific miRNA signatures, and these miRNA signatures were different for the cisplatin vs 5-FU resistant cells from the same tumor cell line, and also for EAC vs ESCC cells with resistance to the same specific chemotherapy agent. Amongst others, miR-27b-3p, miR-193b-3p, miR-192-5p, miR-378 a-3p, miR-125a-5p and miR-18a-3p were dysregulated, consistent with negative posttranscriptional control of KRAS, TYMS, ABCC3, CBL-B and ERBB2 expression via these miRNAs.
CONCLUSION: The current study supports the hypothesis that microRNA expression has an impact on chemotherapy resistance in esophageal cancer.

Chen J, Xu ZY, Wang F
Association between DNA methylation and multidrug resistance in human glioma SHG-44 cells.
Mol Med Rep. 2015; 11(1):43-52 [PubMed] Free Access to Full Article Related Publications
The aim of the present study was to evaluate the association between DNA methylation and multidrug resistance (MDR) in glioma and identify novel effectors responsible for MDR in human gliomas. An MDR glioma cell line, SGH-44/ADM, was developed using adriamycin (ADM) impulse treatment. Cryopreservation, recovery and withdrawal were performed to evaluate the stability of SGH-44/ADM cells. The adherence rate and cellular morphology were observed by microscopy, and the cell growth curve and doubling time were determined. DNA methylation was analyzed using a methylated DNA immunoprecipitation microarray chip (MeDIP-Chip). The cell cycle, Rh123 ingestion and exudation, and SGH-44/ADM apoptosis were analyzed by flow cytometry. SGH-44/ADM cells showed little difference as compared with parental cells, except that SGH-44/ADM cells were bigger in size with a wizened nucleus. Compared to SGH-44 cells, a larger proportion of SGH-44/ADM cells remained in G1 and S phase, as measured by flow cytometry. The MDR of SGH-44/ADM was associated with the upregulation of multi-drug resistance 1, prostaglandin-endoperoxide synthase 2 (COX-2); protein kinase C α (PKCα); however, the expression of these genes was not associated with DNA methylation. In the MeDIP-Chip analysis, 74 functions were markedly enhanced, and seven significant pathways were observed. Genes including SNAP47, ARRB2, PARD6B, TGFB1, VPS4B and CBLB were identified by gene ontology analysis. The predominant molecular mechanism of MDR in SGH-44/ADM cells was identified as exocytosis and efflux. The expression of COX-2, PKCα and P-glycoprotein (Pgp) was not found to be associated with DNA methylation. Genes including SNAP47, VAMP4 and VAMP3 may serve as the downstream effectors of Pgp, COX-2 or PKCα; however, further experiments are required to verify these observations.

Li H, Xu L, Li C, et al.
Ubiquitin ligase Cbl-b represses IGF-I-induced epithelial mesenchymal transition via ZEB2 and microRNA-200c regulation in gastric cancer cells.
Mol Cancer. 2014; 13:136 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Insulin-like growth factor I (IGF-I) can induce epithelial mesenchymal transition (EMT) in many epithelial tumors; however, the molecular mechanism by which this occurs is not clearly understood. Additionally, little is known about the involvement of IGF-I in gastric cancer.
METHODS: Two gastric cancer cell lines were treated with IGF-I to induce EMT and levels of transcription factor ZEB2 and microRNA-200c (miR-200c) were measured. Cells were treated with Akt/ERK inhibitors to investigate the role of these pathways in IGF-I-mediated EMT. Transfection of shRNA plasmids was used to silence the ubiquitin ligase Cbl-b to assess its involvement in this process. The relationship between IGF-IR and Cbl-b expression, and the effect of IGF-IR and Cbl-b on metastasis were analyzed in primary gastric adenocarcinoma patients.
RESULTS: IGF-I-induced gastric cancer cell EMT was accompanied by ZEB2 up-regulation. Furthermore, both Akt/ERK inhibitors and knockdown of Akt/ERK gene reversed IGF-I-induced ZEB2 up-regulation and EMT through up-regulation of miR-200c, suggesting the involvement of an Akt/ERK-miR-200c-ZEB2 axis in IGF-I-induced EMT. The ubiquitin ligase Cbl-b also ubiquitinated and degraded IGF-IR and inhibited the Akt/ERK-miR-200c-ZEB2 axis, leading to the repression of IGF-I-induced EMT. There was a significant negative correlation between the expression of IGF-IR and Cbl-b in gastric cancer patient tissues (r = -0.265, p < 0.05). More of patients with IGF-IR-positive expression and Cbl-b-negative expression were with lymph node metastasis (p < 0.001).
CONCLUSIONS: Together, these findings demonstrate that the ubiquitin ligase Cbl-b represses IGF-I-induced EMT, likely through targeting IGF-IR for degradation and further inhibiting the Akt/ERK-miR-200c-ZEB2 axis in gastric cancer cells.

Hsu TI, Lin SC, Lu PS, et al.
MMP7-mediated cleavage of nucleolin at Asp255 induces MMP9 expression to promote tumor malignancy.
Oncogene. 2015; 34(7):826-37 [PubMed] Related Publications
Nucleolin (NCL) participates in DNA transcription, ribosomal biogenesis and the regulation of RNA stability. However, the contribution of NCL to tumor development is still not clear. Herein, we found that NCL expression correlated with poor prognosis in lung cancer patients. Overexpressed NCL was predominantly cleaved to C-terminal truncated NCL (TNCL). In lung cancer formation, activation of the epidermal growth factor receptor pathway induced NCL expression, and also the expression of matrix metalloproteinase (MMP) 7, which then cleaved NCL at Asp255 to generate TNCL of 55 kDa. TNCL increased the expression of several oncogenes, including MMP9, anaplastic lymphoma kinase (ALK), HIF1a and CBLB, and decreased the expression of tumor suppressors including BRD4, PCM1, TFG and KLF6 by modulating mRNA stability through binding to the 3'-untranslated regions of their transcripts, thus ultimately enhancing metastasis activity. In conclusion, this study identified a novel role of the cleavage form of NCL generated by MMP7 in stabilizing MMP9 mRNA. We also provide a new insight that MMP7 not only cleaves the extracellular matrix to promote tumor invasion but also cleaves NCL, which augment oncogenesis. Blocking NCL cleavage may provide a useful new strategy for lung cancer therapy.

Almog N, Briggs C, Beheshti A, et al.
Transcriptional changes induced by the tumor dormancy-associated microRNA-190.
Transcription. 2013 Jul-Aug; 4(4):177-91 [PubMed] Free Access to Full Article Related Publications
Tumor dormancy is a highly prevalent stage in cancer progression. We have previously generated and characterized in vivo experimental models of human tumor dormancy in which micro-tumors remain occult until they spontaneously shift into rapid tumor growth. We showed that the dormant micro-tumors undergo a stable microRNA (miRNA) switch during their transition from dormancy to a fast-growing phenotype and reported the identification of a consensus signature of human tumor dormancy-associated miRNAs (DmiRs). miRNA-190 (miR-190) is among the most upregulated DmiRs in all dormant tumors analyzed. Upregulation of miR-190 led to prolonged tumor dormancy in otherwise fast-growing glioblastomas and osteosarcomas. Here we investigate the transcriptional changes induced by miR-190 expression in cancer cells and show similar patterns of miR-190 mediated transcriptional reprogramming in both glioblastoma and osteosarcoma cells. The data suggests that miR-190 mediated effects rely on an extensive network of molecular changes in tumor cells and that miR-190 affects several transcriptional factors, tumor suppressor genes and interferon response pathways. The molecular mechanisms governing tumor dormancy described in this work may provide promising targets for early prevention of cancer and may lead to novel treatments to convert the malignant tumor phenotype into an asymptomatic dormant state.

Mu X, Zhang Y, Qu X, et al.
Ubiquitin ligase Cbl-b is involved in icotinib (BPI-2009H)-induced apoptosis and G1 phase arrest of EGFR mutation-positive non-small-cell lung cancer.
Biomed Res Int. 2013; 2013:726375 [PubMed] Free Access to Full Article Related Publications
Epidermal growth factor receptor (EGFR) is one of the most promising targets for non-small-cell lung cancer (NSCLC). Icotinib, a highly selective EGFR tyrosine kinase inhibitor (EGFR-TKI), has shown promising clinical efficacy and safety in patients with NSCLC. The exact molecular mechanism of icotinib remains unclear. In this study, we first investigated the antiproliferative effect of icotinib on NSCLC cells. Icotinib significantly inhibited proliferation of the EGFR-mutated lung cancer HCC827 cells. The IC50 values at 48 and 72 h were 0.67 and 0.07 μ M, respectively. Flow cytometric analysis showed that icotinib caused the G1 phase arrest and increased the rate of apoptosis in HCC827 cells. The levels of cyclin D1 and cyclin A2 were decreased. The apoptotic process was associated with activation of caspase-3, -8, and poly(ADP-ribose) polymerase (PARP). Further study revealed that icotinib inhibited phosphorylation of EGFR, Akt, and extracellular signal-regulated kinase. In addition, icotinib upregulated ubiquitin ligase Cbl-b expression. These observations suggest that icotinib-induced upregulation of Cbl-b is responsible, at least in part, for the antitumor effect of icotinib via the inhibition of phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways in EGFR-mutated NSCLC cells.

Hinterleitner R, Gruber T, Pfeifhofer-Obermair C, et al.
Adoptive transfer of siRNA Cblb-silenced CD8+ T lymphocytes augments tumor vaccine efficacy in a B16 melanoma model.
PLoS One. 2012; 7(9):e44295 [PubMed] Free Access to Full Article Related Publications
The ubiquitin ligase Cbl-b is an established regulator of T cell immune response thresholds. We recently showed that adoptive cell transfer (ACT) of cblb(-/-) CD8(+) T cells enhances dendritic cell (DC) immunization-mediated anti-tumor effects in immune-competent recipients. However, translation of cblb targeting to clinically applicable concepts requires that inhibition of cblb activity be transient and reversible. Here we provide experimental evidence that inhibition of cblb using chemically synthesized siRNA has such potential. Silencing cblb expression by ex vivo siRNA transfection of polyclonal CD8(+) T cells prior to ACT increased T cell tumor infiltration, significantly delayed tumor outgrowth, and increased survival rates of tumor-bearing mice. As shown by ex vivo recall assays, cblb silencing resulted in significant augmentation of intratumoral T cell cytokine response. ACT of cblb-silenced polyclonal CD8(+) T cells combined with DC-based tumor vaccines predominantly mediated anti-tumor immune responses, whereas no signs of autoimmunity could be detected. Importantly, CBLB silencing in human CD8(+) T cells mirrored the effects observed for cblb-silenced and cblb-deficient murine T cells. Our data validate the concept of enhanced anti-tumor immunity by repetitive ACT of ex vivo cblb siRNA-silenced hyper-reactive CD8(+) T cells as add-on adjuvant therapy to augment the efficacy of existing cancer immunotherapy regimens in clinical practice.

Kang JM, Park S, Kim SJ, et al.
CBL enhances breast tumor formation by inhibiting tumor suppressive activity of TGF-β signaling.
Oncogene. 2012; 31(50):5123-31 [PubMed] Related Publications
Casitas B-lineage lymphoma (CBL) protein family functions as multifunctional adaptor proteins and E3 ubiquitin ligases that are implicated as regulators of signaling in various cell types. Recent discovery revealed mutations of proto-oncogenic CBL in the linker region and RING finger domain in human acute myeloid neoplasm, and these transforming mutations induced carcinogenesis. However, the adaptor function of CBL mediated signaling pathway during tumorigenesis has not been well characterized. Here, we show that CBL is highly expressed in breast cancer cells and significantly inhibits transforming growth factor-β (TGF-β) tumor suppressive activity. Knockdown of CBL expression resulted in the increased expression of TGF-β target genes, PAI-I and CDK inhibitors such as p15(INK4b) and p21(Cip1). Furthermore, we demonstrate that CBL is frequently overexpressed in human breast cancer tissues, and the loss of CBL decreases the tumorigenic activity of breast cancer cells in vivo. CBL directly binds to Smad3 through its proline-rich motif, thereby preventing Smad3 from interacting with Smad4 and blocking nuclear translocation of Smad3. CBL-b, one of CBL protein family, also interacted with Smad3 and knockdown of both CBL and CBL-b further enhanced TGF-β transcriptional activity. Our findings provide evidence for a previously undescribed mechanism by which oncogenic CBL can block TGF-β tumor suppressor activity.

Kobashigawa Y, Tomitaka A, Kumeta H, et al.
Autoinhibition and phosphorylation-induced activation mechanisms of human cancer and autoimmune disease-related E3 protein Cbl-b.
Proc Natl Acad Sci U S A. 2011; 108(51):20579-84 [PubMed] Free Access to Full Article Related Publications
Cbl-b is a RING-type E3 ubiquitin ligase that functions as a negative regulator of T-cell activation and growth factor receptor and nonreceptor-type tyrosine kinase signaling. Cbl-b dysfunction is related to autoimmune diseases and cancers in humans. However, the molecular mechanism regulating its E3 activity is largely unknown. NMR and small-angle X-ray scattering analyses revealed that the unphosphorylated N-terminal region of Cbl-b forms a compact structure by an intramolecular interaction, which masks the interaction surface of the RING domain with an E2 ubiquitin-conjugating enzyme. Phosphorylation of Y363, located in the helix-linker region between the tyrosine kinase binding and the RING domains, disrupts the interdomain interaction to expose the E2 binding surface of the RING domain. Structural analysis revealed that the phosphorylated helix-RING region forms a compact structure in solution. Moreover, the phosphate group of pY363 is located in the vicinity of the interaction surface with UbcH5B to increase affinity by reducing their electrostatic repulsion. Thus, the phosphorylation of Y363 regulates the E3 activity of Cbl-b by two mechanisms: one is to remove the masking of the RING domain from the tyrosine kinase binding domain and the other is to form a surface to enhance binding affinity to E2.

Nicholson L, Knight T, Matheson E, et al.
Casitas B lymphoma mutations in childhood acute lymphoblastic leukemia.
Genes Chromosomes Cancer. 2012; 51(3):250-6 [PubMed] Related Publications
Casitas B-lineage lymphoma (CBL) proteins are RING finger ubiquitin E3 ligases that attenuate the signaling of receptor tyrosine kinases and are mutated in a number of myeloid disorders. In this study, mutational screening of the linker-RING domains of CBL and CBLB was performed by denaturing high performance liquid chromatography in a cohort of diagnostic (n = 180) or relapse (n = 46) samples from children with acute lymphoblastic leukemia. Somatic mutations were identified in three children, giving an overall incidence of 1.7% and involved small deletions affecting the intron/exon boundaries of exon 8, leading to skipping of exon 8 and abolishing E3 ligase function. Mutated primary samples were associated with constitutive activation of the RAS pathway and sensitivity to MEK inhibitors was shown. Thus, mutation of CBL is an alternative route to activate the RAS pathway and may identify children who are candidates for MEK inhibitor clinical trials.

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