Molecular Mechanisms of Loss of β2-Microglobulin Expression in Drug-Resistant Breast Cancer Sublines and Its Involvement in Drug Resistance

Abstract

In this study, we investigated the mechanism of the loss or decreased expression of β₂-microglobulin (β₂m) in several drug-resistant sublines of MCF-7 and in a doxorubicin (DOX)-resistant variant of the T-47D breast cancer cell line. β₂m protein and RNA are not expressed in highly metastatic, multidrug-resistant MCF-7/Adr cells with high resistance to DOX. Nuclear run-on transcription and RNA stability assays demonstrate that while β₂m in MCF-7/Adr cells is transcribed, its mRNA is rapidly degraded after synthesis in these cells, indicating that it is controlled by post-transcriptional mechanisms. We also show that an MCF-7 subline (MCF-7/Adr-5) expressing a very low level of resistance to DOX has a decreased level of β₂m expression. Treatment with actinomycin D revealed that the half-life of β₂m mRNA in MCF-7 and MCF-7/Adr-5 cell lines was comparable. Nuclear run-on transcription analysis revealed a decreased rate of β₂m transcription in MCF-7/Adr-5 cells compared to that in MCF-7 cells. Moreover, β₂m mRNA remained undetectable in MCF-7/Adr cells following cycloheximide treatment. However, in MCF-7 cells, increased β₂m mRNA was observed after 12 h, and a similar level of increased mRNA expression was observed after 36 h of cycloheximide treatment in MCF-7/Adr-5 cells; these results suggest that one of the mechanisms controlling β₂m mRNA expression might be a negative regulatory protein in MCF-7/Adr-5 cells. Analysis of the β₂m status of other drug-resistant MCF-7 sublines revealed that deregulation of β₂m is not limited to DOX resistance, but can also be detected in cells selected for resistance to mAMSA and DOX-verapamil. In addition, our data show that reduced β₂m expression correlates with the decreased levels of estrogen receptor (ER) expression in the DOX-resistant MCF-7/Adr and T-47D/Adr-4 human breast cell lines. Furthermore, we provide evidence that the partial inhibition of β₂m by antisense RNA results in 2−3-fold decreased sensitivity of MCF-7 cells to DOX and mAMSA. Moreover, the addition of exogenous β₂m protein near its physiological human serum concentration can modulate the DOX sensitivity of the MCF-7 antisense β₂m and control transfectants. Therefore, these results indicate that lost or decreased β₂m expression is involved in the development of the drug-resistant phenotype and correlates with the loss of ER in human breast cancer cell lines.