The Effects Of Various Recombined Chimeric Cbl Ubiquitin Ligases On The Growth Of Tumour Cells

Cbl(Casitas B-lineage lymphoma), a ubiquitous cytoplasmic protein, belongs to RING-type ubiquitin ligases (or E3). It consists of a conserved N-terminal tyrosine kinase binding (TKB) domain and a RING finger domain in addition to other protein-protein interaction motifs. With the TKB domain and other domains recognizing and interacting with specific substrates while RING finger domain recruiting the ubiquitin-conjugating enzyme E2, Cbl is able to promote ubiquitination and subsequent degradation of its associated proteins. In this manner, Cbl is involved in the negative regulation of signal trusduction and plays an important role in maintaining cell homestasis. It was reported that mutant forms of Cbl may act as oncoproteins; while some oncogenic proteins (for example receptor tyrosine kinase, RTK) related with cancer cell proliferation are able to escape from Cbl-mediated negative regulation, due to their mutations or other genetic changes. The above results highly suggest that negatively regulatory mechanisms of Cbl could be explored to inhibit the growth of cancer cells by controlling early receptorsignals.For the purpose of harnessing Cbl ubiquitin ligase activity to specifically degrade some signal transducer and thus negatively regulate the tumour cell proliferation, we constructed several chimeric Cbl ubiquitin ligases. These chimeric molecules consist of Cbl's N-terminal TKB and RING domains, which are sufficient for its E3 activity, while their SH2 domain within TKB were replaced by SH2 from Grb2, Grb7, p85 or Src, each of them being able to interact with and transmit signals from active EGFR or HER2. We expected these chimeric Cbls, named as Cbl-Grb2(SH2)-RING, Cbl-Grb7(SH2)-RING, Cbl-src(SH2)-RING or Cbl-p85 (SH2)-RING (abbreviated as CblN/Grb2, CblN/Grb7, CblN/p85 or CblN/Src), would specifically promote ubiquitination and degradation of active EGFR or HER2. At the same time, we constructed full length Cbl, Cbl's N-terminal part(CblN) and another U-box E3 ligase CHIP which is capable of down-regulating HER2, as controls. Each of these molecules was cloned into eukaryotic expressing vector pcDNA3.1(+).Firstly, we introduced these chimeric Cbls either into COS-7 cells together with EGFR-expressing plasmids or into EGFR-overexpressing A431 cells, and found that all of them promote EGF-dependent EGFR downregulation, just like wt-Cbl does. This indicates chimeric Cbls have ubiqitin ligase activity.To investigate the effect of chimeric Cbls on the intended substrates and the related biological characteristics of the cells, we transfected the plasmids encoding these molecules into EGFR-positive lung cancer cell line A549, HER2-positive breast cancer cell line SK-BR-3 and EphA2-positive breast cancer cell line MDA-MB-231 respectively. Confirming the expressions of the introduced genes both at transcriptional levels and protein levels, we observed the changes in corresponding receptor levels, the cell growth and other malignant behaviors. We found that: (1)Both Cbl and CblN/Grb7 are able to promote EGF-induced degradation of EGFR and consequently inhibit EGF-stimulated cell proliferation in A549 cells. (2)HER2 protein in SK-BR-3 cells was downregulated by Cbl, CblN/Grb2, CblN/Grb7, CblN/p85, CblN/Src or CHIP in a posttranscriptionalmanner, but not by CblN. Among them, CblN/Grb2 and CblN/p85 seem to be more effective than others. With decreased levels of HER2, these cells exhibited reduced colony-forming ability. (3)In MDA-MB-231 stable clones, only Cbl caused EphA2 downregulation, whereas CblN and CblN/Grb2 did not. Overexpression of Cbl led to inhibition in soft agar colonization, cellular invasiveness, anoikis resistance, MMP-2 secretion, and as expected, suppression of tumor growth in nude mice. In conclusion, wild type Cbl is able to degrade not only active EGFR and HER2, but also EphA2 receptor; while CblN is effective only for EGFR. CblN/Grb2, CblN/Grb7, CblN/p85 and CblN/Src, however, are efficient both for EGFR and HER2. The data presented here indicate that the chimeric Cbls are able to downregulate the targeted proteins and therefore inhibit the tumor cell growth, with effectiveness and specificity to some extent.To further confirm the effect of the chimeric Cbls, we subcloned Cbl/Grb7, Cbl/Src and CHIP into ecdysone-incucible expression vector pIND and transfected them into SK-BR-3 cells stably. In these cells, we proved that ponA treatment induced expression of the transfected genes, therefore led to HER2 downregulation. Consequently, cell growth was inhibited, with G1 cell cycle arrest and decreased colony-forming ability.To examine whether it is through promoting ubiquitination that the chimeric Cbls downregulate the targeted receptors and therefore slow down the cell growth, we performed interaction assay and ubiquitination assay both in vitro and in vivo. Cbl-Grb2(SH2)-RING and CHIP were subcloned into prokaryotic expressing vector pGEX4T-2, and subsequently, GST-Cbl-Grb2(SH2)-RING and GST-CHIP fusion protein were expressed and purified respectively. GST-pull down assay and in vitro ubiquitination asssy showed that the fusion proteins not only interact with HER2, but also mediate autoubiquitination reactions. In vivo co-immuno-precipitation assay demonstrated that overexpressed Cbl, CblN/Grb2, CblN/Grb7, CblN/p85, CblN/Src or CHIP interacts with HER2 in SK-BR-3 cells. Moreover, HER2 ubiquitination was increased in CblN/Grb2 or CHIP-stably transfected SK-BR-3 cells compared with control cells.