CBL Is Frequently Altered in Lung Cancers: Its Relationship to Mutations in MET and EGFR Tyrosine Kinases

Background: Non-small cell lung cancer (NSCLC) is a heterogeneous group of disorders with a number of genetic and proteomic alterations. c-CBL is an E3 ubiquitin ligase and adaptor molecule important in normal homeostasis and cancer. We determined the genetic variations of c-CBL, relationship to receptor tyrosine kinases (EGFR and MET), and functionality in NSCLC. Methods and Findings: Using archival formalin-fixed paraffin embedded (FFPE) extracted genomic DNA, we show that c-CBL mutations occur in somatic fashion for lung cancers. c-CBL mutations were not mutually exclusive of MET or EGFR mutations; however they were independent of p53 and KRAS mutations. In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH) for the c-CBL locus (22%, n = 8/37) and none of these samples revealed any mutation in the remaining copy of c-CBL. The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples. Using select c-CBL somatic mutations such as S80N/H94Y, Q249E and W802* (obtained from Caucasian, Taiwanese and African-American samples, respectively) transfected in NSCLC cell lines, there was increased cell viability and cell motility. Conclusions: Taking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis

Genome-wide association study identifies human genetic variants associated with fatal outcome from Lassa fever

Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many individuals. Here, to investigate whether human genetic variation underlies the heterogeneity of LASV infection, we carried out genome-wide association studies (GWAS) as well as seroprevalence surveys, human leukocyte antigen typing and high-throughput variant functional characterization assays. We analysed Lassa fever susceptibility and fatal outcomes in 533 cases of Lassa fever and 1,986 population controls recruited over a 7 year period in Nigeria and Sierra Leone. We detected genome-wide significant variant associations with Lassa fever fatal outcomes near GRM7 and LIF in the Nigerian cohort. We also show that a haplotype bearing signatures of positive selection and overlapping LARGE1, a required LASV entry factor, is associated with decreased risk of Lassa fever in the Nigerian cohort but not in the Sierra Leone cohort. Overall, we identified variants and genes that may impact the risk of severe Lassa fever, demonstrating how GWAS can provide insight into viral pathogenesis

Enigma prevents Cbl-c-mediated ubiquitination and degradation of RETMEN2A.

The Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a highly conserved family of RING finger ubiquitin ligases (E3s) that function as negative regulators of tyrosine kinases in a wide variety of signal transduction pathways. In this study, we identify a new Cbl-c interacting protein, Enigma (PDLIM7). This interaction is specific to Cbl-c as Enigma fails to bind either of its closely related homologues, Cbl and Cbl-b. The binding between Enigma and Cbl-c is mediated through the LIM domains of Enigma as removal of all three LIM domains abrogates this interaction, while only LIM1 is sufficient for binding. Here we show that Cbl-c binds wild-type and MEN2A isoforms of the receptor tyrosine kinase, RET, and that Cbl-c enhances ubiquitination and degradation of activated RET. Enigma blocks Cbl-c-mediated RETMEN2A ubiquitination and degradation. Cbl-c decreased downstream ERK activation by RETMEN2A and co-expression of Enigma blocked the Cbl-c-mediated decrease in ERK activation. Enigma showed no detectable effect on Cbl-c-mediated ubiquitination of activated EGFR suggesting that this effect is specific to RET. Through mapping studies, we show that Cbl-c and Enigma bind RETMEN2A at different residues. However, binding of Enigma to RETMENA prevents Cbl-c recruitment to RETMEN2A. Consistent with these biochemical data, exploratory analyses of breast cancer patients with high expression of RET suggest that high expression of Cbl-c correlates with a good outcome, and high expression of Enigma correlates with a poor outcome. Together, these data demonstrate that Cbl-c can ubiquitinate and downregulate RETMEN2A and implicate Enigma as a positive regulator of RETMEN2A through blocking of Cbl-mediated ubiquitination and degradation

Cbl-c ubiquitin ligase activity is increased via the interaction of its RING finger domain with a LIM domain of the paxillin homolog, Hic 5.

Cbl proteins (Cbl, Cbl-b and Cbl-c) are ubiquitin ligases that are critical regulators of tyrosine kinase signaling. In this study we identify a new Cbl-c interacting protein, Hydrogen peroxide Induced Construct 5 (Hic-5). The two proteins interact through a novel interaction mediated by the RING finger of Cbl-c and the LIM2 domain of Hic-5. Further, this interaction is mediated and dependent on specific zinc coordinating complexes within the RING finger and LIM domain. Binding of Hic-5 to Cbl-c leads to an increase in the ubiquitin ligase activity of Cbl-c once Cbl-c has been activated by Src phosphorylation or through an activating phosphomimetic mutation. In addition, co-transfection of Hic-5 with Cbl-c leads to an increase in Cbl-c mediated ubiquitination of the EGFR. These data suggest that Hic-5 enhances Cbl-c ubiquitin ligase activity once Cbl-c has been phosphorylated and activated. Interactions between heterologous RING fingers have been shown to activate E3s. This is the first demonstration of enhancement of ubiquitin ligase activity of a RING finger ubiquitin ligase by the direct interaction of a LIM zinc coordinating domain

Enigma abrogates RETMEN2A ubiquitination.

<p>(<b>A</b>) RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates described above and immunoblotted for HA-ubiquitin and phosphotyrosine (pY) as indicated on the right. (<b>B</b>) HEK293T cells were transfected with GST-Cbl-c with and without FLAG-Enigma along with either RET9MEN2A or RET51MEN2A. Cells were collected 48h post-transfection, and whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates and immunoblotted for RET and HA-ubiquitin as indicated on the right. Molecular weight standards are indicated on the left and Ponceau S staining serves as a measure of protein loading. (<b>C</b>) HEK293T cells were transfected with RET9MEN2A with GST-Cbl-c and FLAG-Enigma both alone and in combination as indicated above the blots. All transfections were performed in triplicate. At 24h post-transfection, triplicate plates were pooled and replated. At 40h post-transfection, replicate plates were treated with 100ng/mL cycloheximide (CHX) for either 3 or 5h as indicated. Control plates received an equivalent volume of DMSO vehicle control for 5h prior to cell collection. A total of 20 µg of each of the whole cell lysates was immunoblotted as indicated. RET steady state levels were then assessed using β-actin as a loading control. (<b>D</b>) Densitometric analysis of RET steady state levels in the presence of GST-Cbl-c and Enigma, either alone or in combination. Levels were all compared to Vector transfected cells in the absence of cycloheximide. Error bars denote mean ± SE (n  = 3). (<b>E</b>) HEK293T cells were transfected with RET9MEN2A with GST-Cbl-c and FLAG-Enigma both alone and in combination as indicated above each blot. At 24h post-transfection, all cells were starved of FBS for 24h prior to cell harvesting. A total of 20 µgs of each of the whole cell lysates were immunoblotted as indicated to the right of each panel. Phospho-MAPK steady state levels were assessed using MAPK and Hsc70 as loading controls. Molecular weight standards are indicated on the left of each panel. (<b>F</b>) HEK293T cells were transfected with wild-type RET and the RET co-receptor, GFRα1 along with GST-Cbl-c and Enigma both alone and in combination. Vector controls included empty GST vector and all transfections were performed in duplicate and included HA-tagged ubiquitin. At 24h post-transfection, duplicate plates were pooled and re-plated to allow reattachment overnight. At 40h post-transfection, plates were rinsed and starved in media lacking FBS for 24h, then stimulated as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087116#pone.0087116-Boulay1" target="_blank">[43]</a>, using 30ng/mL GDNF and 100ng/mL exogenous GFRα1 (+) or water control (-) for 15m as indicated prior to collection and lysis. Whole cell lysates were immunoblotted as indicated. To assess RET ubiquitination, RET immunoprecipitations were performed on 300ugs of whole cell lysate and immunoblotted for HA-ubiquitin as indicated on the right. (<b>G</b>) HEK293T cells were transfected with EGFR along with GST-Cbl-c and Enigma both alone and in combination. All transfections were performed in duplicate. At 24h post-transfection, duplicate plates were pooled and replated to allow reattachment overnight. At 40h post-transfection, plates were rinsed and starved in media lacking FBS for 8h, then stimulated with 100ng/mL EGF (+) or water control (-) for 10m as indicated prior to collection and lysis. Whole cell lysates were immunoblotted as indicated. To assess EGFR ubiquitination, EGFR immunoprecipitations were performed on 300 µg of whole cell lysate and immunoblotted for EGFR and HA-ubiquitin as indicated on the right. Molecular weight standards are shown to the left of each panel and tubulin serves as loading control.</p

Enigma blocks Cbl-c-mediated RET9MEN2A ubiquitination.

<p>(<b>A</b>) HEK293T cells were transfected with RET9MEN2A along with GST-Cbl-c, FLAG-Enigma or FLAG-EnigmaΔLIM2-3 alone and in combination as indicated above the blots. All transfections included an equal amount of HA-ubiquitin. At 40h post-transfection, all cells were treated with 20nM MG-132 for 5h prior to cell collection. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of whole cell lysate and immunoblotted for RET and HA-ubiquitin. Co-immunoprecipitation of Cbl-c and Enigma were evaluated as indicated on the right. Molecular weight standards are indicated to the left of each panel and Hsc70 serves as a loading control. (<b>B</b>) RET truncation constructs used for mapping Cbl-c and Enigma interaction sites. Structural domains include the extracellular cadherin-like repeats (C1-4), a cysteine rich region (CR), a hydrophobic transmembrane region (TM), a split cytoplasmic tyrosine kinase domain (TK1 and TK2), and intracellular tyrosines subsequently mutated to an F or stop (*) to create each construct used in this study. (<b>C</b>) HEK293T cells were transfected with GFP-tagged Enigma both alone and in combination with RET9MEN2A or one of a series of RET9MEN2A mutant constructs as indicated above each blot. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates and immunoblotted for GFP-Enigma. Molecular weight standards are indicated on the left of each panel and Hsc70 serves as loading control. (<b>D</b>) HEK293T cells were transfected with each of the RET9MEN2A constructs both alone and in combination with GST-Cbl-c as indicated above each blot. All transfections were balanced with empty vector controls, and cells were collected 48 h post-transfection. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. GST pull-downs were performed on 300 µg of each of the whole cell lysates and immunoblotted for Cbl-c, phosphotyrosine (pY), and RET as indicated on the right. Molecular weight standards are indicated to the left of each panel and Hsc70 serves as a loading control.</p

Cbl binds RET and MEN2A kinases.

<p>(<b>A</b>) HEK293T cells were transfected separately with pJ7Ω plasmids encoding each of the following four RET kinase isoforms: RET 9, RET 51, and their MEN2A (C634R) mutant isoforms. Each was transfected both with and without GST-Cbl-c. Whole cell lysates were collected at 48h post-transfection and GST pull-downs were performed on 300 µg of whole cell lysates and immunoblotted (IB) as indicated. Whole cell lysates were immunoblotted as indicated and molecular weight standards are indicated on the left of each panel β-actin serves as a loading control. (<b>B</b>) HEK293T cells were transfected with GST-Cbl-c both with and without RET9MEN2A. Replicate plates were treated with either 500 µM Sorafenib (+) or DMSO as vehicle control (-) for 90m prior to collection. Whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of whole cell lysates using anti-RET antibody with Protein A/G Sepharose and immunoblotted for RET and phosphotyrosine (pY) as indicated. GST pull-downs were performed, as described above, and immunoblotted (IB) for RET, phosphotyrosine (pY), and GST-Cbl-c as indicated. Molecular weight standards are indicated to the left of each panel and Hsc70 serve as loading controls.</p

Loss of function Cbl-c mutations in solid tumors.

Receptor Tyrosine Kinase (RTK) signaling is essential for normal biological processes and disruption of this regulation can lead to tumor initiation and progression. Cbl proteins (Cbl, Cbl-b and Cbl-c) are a family of RING finger (RF) ubiquitin ligases that negatively regulate a variety of RTKs, including EGFR, MET, and RET. Recent studies have identified Cbl mutations associated with human myeloid neoplasias in approximately 5% of the cases. Cbl-c is the most recently identified human Cbl protein and is expressed exclusively in epithelial cells. We identified a novel cDNA that was isolated from a mouse mammary cancer from the C3(1) Large T Antigen transgenic model. This mutant cDNA encodes a protein that has a deletion in the RF domain of Cbl-c, thereby resembling known Cbl family mutations associated with myeoloid neoplasias. Genomic analysis of both parental and transgenic lines shows no evidence of germline mutation indicating that this mutation is likely a somatic mutation. The mutant protein enhances transformation of NIH 3T3 cells when expressed in combination with SV40 Large T antigen. Together these data are consistent with a second hit mutation. In overexpression studies, this mutant Cbl-c protein fails to mediate ubiquitination of activated EGFR and acts in a dominant negative fashion to prevent ubiquitination and downregulation of the activated EGFR by wild type Cbl proteins. Mechanistically, the mutant Cbl-c binds to the EGFR and prevents recruitment of the wild type Cbl protein. Furthermore, data mining reveals Cbl-c mutations associated with solid tumors in humans. Subsequent cell-based analysis demonstrates a similar loss of E3 function and dominant negative effects for one of these human mutations. These data suggest that like Cbl mutations in myeloid neoplasms, loss of Cbl-c function may contribute to the pathogenesis of solid tumors in murine models and in humans