Identification of heat shock protein 90α as an IMH-2 epitope-associated protein and correlation of its mRNA overexpression with colorectal cancer metastasis and poor prognosis.

PURPOSE: We studied whether HSP90α was associated with the special carbohydrate structures IMH-2 epitopes, and investigated its mRNA expression and clinical relevance in colorectal cancer (CRC) patients.
METHODS: The lysates and the culture media of colon cancer HCT-8 cells were immunoprecipitated with IMH-2 antibody, and the immunoprecipitates were subsequently analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or by immunoblotting with anti-HSP90α antibody. In vitro wound-healing assay was done to evaluate the role of IMH-2 epitope-associated HSP90α in HCT-8 cell migration. Real-time RT-PCR was performed to detect the levels of HSP90α mRNA expression in paired tumor and non-tumor tissues of 56 CRC patients. The correlation of tumor HSP90α mRNA overexpression with CRC metastasis and poor survival outcome was determined by statistical analyses.
RESULTS: HSP90α was first identified as an IMH-2 epitope-associated protein by immunoprecipiation, mass spectrometry, and immunoblotting analysis. IMH-2 epitopes were detected in both cellular and secreted HSP90α. HCT-8 cell migration induced by serum starvation-conditioned medium was blocked by anti-HSP90α antibody or the HSP90α inhibitor geldanamycin (GA) as efficient as by IMH-2 antibody, suggesting that IMH-2-associated HSP90α was involved in serum starvation-induced CRC cell migration. On the other hand, HSP90α mRNA expression was induced in HCT-8 cells after serum starvation. Clinically, 15 (26.8%) of 56 CRC patients exhibited tumor HSP90α mRNA overexpression and had higher rates of metastatic occurrence (P = 0.003) and poor prognosis (P = 0.028).
CONCLUSIONS: HSP90α was an IMH-2 epitope-associated protein. Tumor HSP90α overexpression was correlated with the metastasis and poor prognosis of CRC patients.