Abstract
Abstract Major histocompatibility complex (MHC) class I molecules play an important role in eliciting host tumor-specific immune response since they present tumor antigen-derived peptides to CD8+ cytotoxic T lymphocytes. Accordingly, loss or downregulaton of MHC class I expression in tumor cells represents an immune escape mechanism utilized by a variety of tumor cell types including breast carcinoma. In this study, we have examined the mechanisms underlying MHC class I loss by the human breast carcinoma cell line MDA-MB-157, which was derived from a metastatic medulallary breast carcinoma lesion. Flow cytometry did not detect MHC class I expression on MDA-MB-157 cells but showed robust expression on MDA-MB-231 cells, a positive-control cell line. Western blot analysis of various antigen processing machinery components and MHC class I subunits in the presence and absence of IFN-□ stimulation identified loss of□□2–microglobulin (□2m), a 12 kDa subunit critical for MHC class I assembly and transport, as a major defect in MDA-MB-157 cells. Subsequently, quantitative real-time RT-PCR and nucleotide sequencing analysis of the □2m mRNA in MDA-MB-157 cells detected fair amounts of steady-state □2m transcripts with no mutations in the coding region, suggesting a post-transcriptional/translational defect. Treatment of MDA-MB-157 cells with the proteasome inhibitor MG-132 did not restore □2m expression, ruling out accelerated □2m protein degradation as a possible mechanism. To investigate whether mutations exist in the □2m mRNA untranslated regions (UTR), Rapid Amplification of cDNA Ends (RACE) was employed but, surprisingly, did not detect any UT fragments compared to controls. Moreover, genomic PCR with walking primers failed to amplify a large internal region of the □2m gene in MDA-MB-157 cells except exon 1 and exon 4. The critical role of □2m in the proper assembly and surface expression of MHC class I was indicated by the restoration of HLA class I on MDA-MB-157 cells stably transfected with a □2m-encoding plasmid. Lastly, cytotoxicity assay demonstrated that MDA-MB-157 cells were resistant to NK cell-mediated cytolysis, suggesting the development of tumor NK-resistance in the absence of HLA class I expression. Taken together, our results revealed novel genetic and post-transcriptional mechanisms underlying total HLA class I loss by a breast carcinoma cell line and its NK-resistance phenotype. These findings may suggest new therapeutic strategies to increase the susceptibility of tumor cells to immune destruction in patients.