Our results demonstrate that c-CBL is somatically mutated (or has LOH) in lung cancers, and can significantly contribute to enhanced cell viability and motility. There was also a high prevalence of LOH with respect to c-CBL in lung tumors that harbored MET or EGFR mutation.
In the present study, we have demonstrated the occurrence of c-CBL mutations in lung cancer patients, especially with different ancestral variations. Mutations in c-CBL have been recently reported in juvenile myelomonocytic leukemia and myeloid malignancies. In the AML study, the mutation R420Q located in the junction of the RING finger and the linker region inhibited FMS-like tyrosine kinase 3 (FLT3) internalization and ubiquitination 
, thus contributing to the gain-in-function for the RTK. In addition, mutations such as H398Y, C384R, and L380P mapped to the RING finger domain and the linker region of c-CBL that is required for its E3 activity 
. Additionally, homozygous mutations in the RING finger domain of the c-CBL
gene were described as a result of acquired Uniparental Disomy (UPD) 
. It is important to note that our results indicate LOH at 11q23 locus and these are mutually exclusive from missense mutations of c-CBL
. The somatic mutations were all heterozygous. The mutations in AML led to abrogation of the E3 activity leading to prolonged RTK activation. In addition mutants located on the linker region surrounding the RING finger domain exhibited enhanced AKT signaling in response to cytokine stimulation 
. In addition, it was shown in NH3T3 cells, that neither mutations in the RING finger nor the linker region causes transformation, however while certain mutations perturbs the ubiquitination, others affect receptor recycling and prolong kinase activity 
We report here c-CBL mutations that mapped not only to the RING finger domain, but also to the TKB domain, proline-rich domain and the C-terminal region, but none mapped to the linker region as reported in the AML studies described above 
. In addition, 8 mutants that we detected were found in different ethnic backgrounds. For example, S80N/H94Y, Q249E, W802* were detected in Caucasians, Taiwanese and African-Americans, respectively. The results point out not only the difference between lung cancer and other cancers, but also genetic polymorphism among different races in the same cancer. Interestingly, there is a large disparity between African-American and other ethnic populations with lung cancer 
. We have previously shown that there was a low frequency of EGFR and MET mutation in African-Americans as compared with Taiwanese and Caucasians 
. In this study, the number of African-American samples analyzed was relatively fewer and we found 3 mutations that are unique to this ethnicity. It would now behoove us to further study the genetic alterations that can occur and determine the targeted therapeutics for African-Americans. Our results provide evidence of the importance of c-CBL in tumorigenesis and potential signaling. Our prediction, based on the AML data, would be that the V391I RING finger domain mutation would affect the E3 activity. Also, it will be important to determine the binding partners for c-CBL in the TKB domain and proline rich domain mutations. It has previously been shown that the TKB domain can bind to growth factor receptors and it will be important to determine the cross-binding of these mutants to MET and or EGFR. It would also be important in the future to look at fluorescence in situ hybridization/copy number changes in c-CBL
in lung cancer.
c-CBL plays an important role in down regulating RTK-mediated signaling through K63 poly-ubiquitination and subsequent downregulation of RTKs followed by lysosomal degredation 
. Mono-ubiquitination or ubiquitinated with K63-linked chains of substrates by c-CBL may lead to enhancement of biological and biochemical functions (reviewed in Hermann et al, 2007 
). The mutations that we analyzed in our studies all point out to the fact that the E3 activity of c-CBL on EGFR is intact; the EGFR levels in the various mutants remain same (Figure S2
). Multiple kinases, both RTKs and non-RTKs could be acted upon by c-CBL, including ERBs, PDGFR, FMS, MET, c-Kit, VEGFR, FLT-1, RON, FGFR, IR, as well as SYK, FYN, LCK, FGR, LYN and c-ABL 
. In lung cancers the relevant substrates of c-CBL in terms of degradation or signal transduction are yet to be identified.
The observation that c-CBL somatic mutations, especially S80N/H94Y, Q249E and W802* showed increased cell viability and cell motility that is in agreement with the physiological role for D-cbl in the regulation of apoptosis and differentiation identified in Drosophila is very significant 
. It has been previously shown that activating c-CBL mutation downregulates EGFR signaling and decreases cellular proliferation and migration in breast cancer cell lines 
. Although the role of c-CBL in the negative regulation of RTKs is well substantiated, thereby suggesting that it is a natural tumor suppressor, studies in cancer cells have revealed both tumor suppressor and tumor promoting activities depending on the type of c-CBL mutation and the number of alleles at the c-CBL
. In agreement with the above, the three c-CBL mutants described here appear to have tumor growth and metastasis promoting properties. Although these mutants are outside of the RING finger or the linker region of c-CBL, their downstream effects are significant so as to cause increased proliferation and migration, but the substrate affected by these mutations are not known yet. This raises the possibility that some of the cellular functions of c-CBL are independent of its ubiquitin-ligase activity, an area that we are currently investigating. The oncogenic nature of RTKs, addiction of cancers to growth signals and given the clustering of c-CBL
mutations, it is possible that the transforming effect of c-CBL mutations is most likely a combinatorial effect of the three. We also show that LOH for c-CBL
was found in a significant number of samples that harbored MET
mutations. The fact that about 7% of lung tumor samples are likely to have c-CBL mutations and an additional 22% are likely to harbor c-CBL
-related LOH, makes c-CBL a highly mutated molecule in lung cancer. Since LOH alone is not enough to cause a transforming event 
, associated mutation in the MET
locus or yet another RTK discussed above may play a role in carcinogenesis. We predict that this LOH in c-CBL
results in haploinsufficiency that downplays RTK ubiquitination leading to hyperactivity of the RTKs. However, whether this is sufficient cause for tumorigenesis remains to be determined. Consistent with our hypothesis is the fact that c-cbl-/-
mice have increased kinase activity in lymphocytes, but is not sufficient for tumor formation 
LOH could also lead to increased expression of c-CBL from the other allele to compensate for a loss of an allele. Alternately, there could be some form of synergy working with reduced c-CBL levels and mutated receptors that exacerbate the phenotype of each alone.
Previous studies from our lab and others have shown that East Asians with lung cancers have relatively high frequencies of gain-of-function of mutations in RTKs such as EGFR and MET 
. In a cohort of Japanese patients an activating MET mutation was identified in the splice region that deletes the juxtamembrane domain that is involved E3 activity of c-CBL 
. This study also found that activation of MET is mutually exclusive of EGFR, KRAS and HER2 gene mutations 
. We failed to detect such mutations in significant numbers in lung tumor samples obtained from African-Americans (n
29) and Caucasian (n
50) patients. One MET mutation was identified in each of the groups whereas 1 and 3 EGFR mutations were identified in the African-American and Caucasian cohorts respectively. EGFR mutations have earlier been identified as one of the key mutations affecting lung adenocarcinoma patients in a comprehensive study of 188 patients 
. Our study encompasses different histologies of NSCLC. However, the published series did not find any mutations in c-CBL
unlike our study that encompassed different subtypes of NSCLC. It is important to note, we have recently shown that MET mutations in lung cancer are in majority germline 
. We have reported earlier c-CBL mutations in a small cohort of Taiwanese lung cancer samples 
. In our efforts to understand the ethnic differences in the lung oncogenome, we also looked at PAX transcription factors such as PAX5 and PAX8 that are highly expressed in lung cancers; however there was no preferential expression or mutations of the above genes in lung tumor samples of African-Americans. In this study, we show relatively high frequency of c-CBL mutations in lung cancers, especially in the large cell type among Caucasians and particularly among African-Americans. We therefore propose c-CBL as an efficacious target for lung cancers in African-Americans that needs to be further substantiated. This is all the more important because the prognosis for African-Americans with lung cancer, especially for men is much poorer compared to their Caucasian counterparts .
In conclusion, the results presented in this study demonstrate that c-CBL is frequently mutated or even lost in lung cancers. Our results support a role for c-CBL mutants that are independent of its ubiquitination activity. Given the relatively high mutation rates in c-CBL as well as RTKs such as MET and EGFR, it is likely that their combined effect could be synergistic in promoting tumorigenesis.