Accelerated HER-2 degradation enhanced ovarian tumor recognition by CTL. Implications for tumor immunogenicity

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelia l cancers. HER-2 degradation was investigated in the ovarian tumor line, SK OV3.A2, that constitutively overexpressed long-life HER-2. We used as agoni st geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence t han in the absence of GA. GA did not decrease HLA-A2 expression. Presentati on of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369-377 ) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation bu t have different effects. Enhanced tumor recognition was not an immediate o r early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK c ell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER- 2 protein synthesized in the presence of GA, of faster electrophoretic mobi lity than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twen ty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity dire cted to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cel ls compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Incre ased HER-2 CTL epitopes presentation may have implications for overcoming t he poor immuno-genicity of human tumors, and design of epitope precursors f or cancer vaccination.