Cyclin D1 Overexpression and Response to Bortezomib Treatment in a Breast Cancer Model

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Abstract
Background: Cyclin D1 is frequently overexpressed in breast cancer, and its overexpression is, surprisingly, associated with improved survival. One potential mechanism for this association involves signal transducer and activator of transcription 3 (STAT3). Methods: Cyclin D1 and STAT3 expression were assessed in human tumors using microarray analysis and in breast cancer cell lines HBL100, T47D, MCF7, MDA-MB-453, and BT20 and in HBL100 and T47D cells stably overexpressing cyclin D1 using immunoblot analysis. Cyclin D1 protein was stabilized by treatment with the proteasome inhibitor bortezomib, and the effects on STAT3 expression in vitro was determined by using immunoblotting and on xenograft tumor growth and apoptosis in vivo was determined by using terminal deoxyuridine nick-end labeling assays. All statistical tests were two-sided. Results: Tumors with high cyclin D1 expression (n = 17) had low STAT3 expression (mean = 274 arbitrary units), and those with low cyclin D1 expression (n = 31) had high STAT3 expression (mean = 882 arbitrary units) ( P <.001). In HBL100 and T47D parental and cyclin D1–overexpressing cells, cyclin D1 overexpression was also inversely associated with STAT3 expression, and cyclin D1 directly reduced the expression of STAT3. Stabilization of cyclin D1 protein by bortezomib treatment further amplified the cyclin D1–dependent repression of STAT3 in vitro and slowed tumor growth in vivo (week 7: untreated mean = 185.7 mm 3 versus treated mean = 136.2 mm 3 , difference = 49.5 mm 3 , 95% confidence interval [CI] = 18 to 81 mm 3 , P = .007; week 8: untreated mean = 240.2 mm 3 versus treated mean = 157.3 mm 3 , difference = 82.9 mm 3 , 95% CI = 9.1 to 156.7 mm 3 , P = .0014; and week 9: untreated mean = 256.4 mm 3 versus treated mean = 170.2 mm 3 , difference = 86.2 mm 3 , 95% CI = 22.8 to 149.6 mm 3 , P = .006) and increased apoptosis (untreated mean = 19% versus treated mean = 54%, difference = 35%, 95% CI = 24.7% to 45.4%; P = .013) of xenograft tumors. Conclusions: Cyclin D1 repression of STAT3 expression may explain the association between cyclin D1 overexpression and improved outcome in breast cancer. In addition, bortezomib can amplify the proapoptotic function of cyclin D1, raising the possibility that cyclin D1 levels may be a marker for predicting the response to this novel drug.