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Abstract

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation 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 but have different effects. Enhanced tumor recognition was not an immediate or 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 cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility 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. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.

Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

“…In contrast to iHsp90 impact on Hsp90 client proteins, the Western blot data show that the observed augmentation of melanocyte differentiation antigens in response to iHsp90 results from increased levels of full-length protein, a further indication that Melan-A/MART-1, gp100 and TRP-2 are not Hsp90 client proteins. These observations are in contrast to previous reports of iHsp90 drugs that lead to antigen degradation of Hsp90 client proteins, such as Her-2 [22] and EphA2 [23] . While the mechanism for antigen augmentation by iHsp90 remains to be determined, the data are consistent with transcriptional regulatory mechanisms, rather than a direct impact of iHsp90 on the antigens themselves.…”

“…However, there is also evidence that iHsp90s can enhance anti- tumor immunity. The degradation of EphA2 [23] , and Her-2 [22] , leads to enhanced recognition by CTL, probably as a result of increased turnover and expression on MHC Class I antigens. The augmented recognition of tumor cells we describe is a result of a true increase in tumor associated antigen expression.…”

“…Notably, iHsp90s have been shown to increase T cell recognition of both Her-2 [22] and EphA2 [23] antigens. Both of these onco-proteins are known client proteins of Hsp90, and while the levels of intracellular expression of these antigens were decreased after Hsp90 treatment, the enhanced CTL-recognition of the treated tumor cells was attributed to increased turnover of the proteins, combined with augmented peptide presentation on MHC molecules.…”

Major Histocompatibility Complex Modulation and Loss

“…The abrogation of CTL epitope processing could also be attributable to single amino acid substitutions in transforming genes [56,57]. Thus, the expression pattern of proteasome subunits in tumors might tune the generation of antigenic peptides as well as the sensitivity to CTL-mediated lysis which could be further enhanced by accelerated degradation of TAAs [56,58]. These alterations may have important implications on the success of T cell-based immunotherapies.…”

Photodynamic Therapy with Hypericin Improved by Targeting HSP90 Associated Proteins

“…HER2 receptor is one of the most sensitive client proteins to geldanamycin and its derivatives which was shown by many experimental and clinical studies [11-17]. …”