Disruption of SPRY2-Cbl interaction inhibits proliferation and EGFR-ERK signaling in HEK293T cells.

C88

Introduction: SPRY2, an endogenous inhibitor of RTK-MAPK signaling, is a tumor suppressor that is most commonly inactivated in cancer cells by epigenetic silencing (promoter CpG methylation), resulting in reduced cellular levels. SPRY2 is in dynamic equilibrium with Cbl, an E3 ubquitin ligase targeting SPRY2 for proteolysis via interaction with its ring finger domain; Cbl also targets EGFR for internalization and degradation. We therefore tested the hypothesis that a competitive peptide inhibitor targeting the Cbl ring finger domain blocks SPRY2 degradation and restores SPRY2 suppressor activity in HEK-293T cells. Methods: HEK-293T cells were primarily used, a model where the biology of SPRY2, Cbl, EGFR, and MAPK signaling are well-defined. SPRY2 protein levels were also assayed (immunoblot) in the NCI-60 cell line panel. cDNA for the peptide competitor, corresponding to AA 36-53 of SPRY2 (nucleotides 478-549, GenBank#AF03983), was cloned into pCDNA3.1; pCDNA3.1- SPRY2hisV5 was a positive control, and pcDNA3.1 (no insert or lacZ) was a negative control. Proliferation of HEK-293T cells was quantified by cell count (Coulter and Guava instruments), in the presence or absence of serum ± EGF ligand. Anchorage-independent growth was quantified by soft agarose clonogenic assay. Total-and phospho-protein levels were quantified by immunoblotting. SPRY2-Cbl complex formation was assayed following immunoprecipitation, and the elimination kinetics of SPRY2 and Cbl proteins were quantified post-cyclohexamide. Results: SPRY2 protein was detected in all but 4 cell lines in the NCI-60 panel, confirming that the protein is generally present in cancer cells. Transiently transfected peptide competitor disrupted immunoprecipitatable SPRY2-Cbl complexes. The competitor inhibited proliferation of HEK-293T cells by at 48-72h post-transfection by 40-70%. Effects of the competitor on anchorage-independent growth are pending. Under normal growth conditions, transient expression of the competitor markedly reduced p-ERK and relative levels of p-EGFR (p1068 and pSer84 versus total). The competitor also slightly reduced AKT pTyr308 but not pSer473. Following serum starvation and EGF ligand (100 ng/ml), transient expression of the competitor was associated with increased p-ERK (as expected - in this model, SPRY2 titrates Cbl and enhances EGF ligand-mediated signaling). Data on the effects of the competitor on elimination kinetics of endogenous SPRY2 are pending. All observed effects of the competitor were similar to those seen with transient over-expression of full-length SPRY2. SPRY2 also potentially modulates senescence and apoptosis, in addition to proliferation; effects of the competitor on SA-βgal and annexin V staining, and levels of p53, Rb, pRb, p16, and PARP (cleaved versus intact) are being tested, and are pending. Conclusions: An inhibitor of SPRY2-Cbl interaction inhibited proliferation and EGFR-to-ERK signaling in HEK-293T cells, mimicking the effects of overexpressed exogenous SPRY2. These data are consistent with enhancement of the activity of endogenous SPRY2, and suggest that targeting SPRY2-Cbl complexes may have potential therapeutic utility (Support: NIH K24CA084081).