QS287. HER2 Regulates Expression of Low Molecular Weight Cyclin E Isoforms in Breast Cancer

Abstract

Objective: HER2 amplification and cyclin E overexpression have each been shown to be important prognostic indicators in breast cancer. In addition to overexpressing the full length (FL) cyclin E protein, some breast cancer cell lines and human breast cancers express up to 5 low molecular weight (LMW) isoforms which are generated by proteolytic processing of FL cyclin E by the serine protease elastase. These LMW forms have been demonstrated to be more tumorigenic than the FL protein. We hypothesized that HER2 overexpression may modulate cyclin E regulation in breast cancer, including generation of the LMW forms. Methods: HER2 expression was downregulated in the HER2-overepressing breast cancer cell lines MCF-7-HER-18 (HER18), SKBr3 and BT474 using 2 different mechanisms; treatment with trastuzumab dosed at 10 and 20 μg/ml or transfection with HER2 siRNA. The low-HER2 expressing cell line MCF-7 was used as a negative control. The effect on cyclin E expression was investigated using confocal immunofluorescence microscopy and western blot analysis. The effect of HER2 downregulation on cyclin E transcription was assessed using quantitative RT-PCR (qRT-PCR). To determine if there may be a clinically relevant relationship between these 2 proteins, HER2 and LMW cyclin E levels were assessed in 25 patient samples collected as part of an ongoing prospective protocol. Results: Confocal immunofluorescence microscopy demonstrated that HER2 downregulation using siRNA or inactivation by treatment with trastuzumab resulted in decreased total cyclin E expression. Because cyclin E is tightly regulated at the level of transcription, qRT-PCR was performed to determine the effects of HER2 downregulation on cyclin E mRNA synthesis. There was no difference (p=.18) in cyclin E mRNA synthesis for cells transfected with HER2 siRNA versus those transfected with random sequence and mock transfected controls suggesting that HER2 affects post-transcriptional processing of cyclin E. Having shown that the effect of HER2 on cyclin E expression is not at the level of transcription, western blot analysis was used to investigate the effects of HER2 downregulation on the LMW forms. Western blot demonstrated that the decrease in cyclin E was primarily in the LMW forms (figure) confirming the effect of HER2 to be post-transcriptional. Western blot analysis was then performed on lysates obtained from 25 patient samples. Nine (35%) patients had HER2-overexpressing tumors. Densitometry was used to quantify FL and LMW cyclin E expression. There was no significant difference in expression of FL cyclin E in HER2+ versus HER2- tumors (p=.87). In contrast, there was significantly higher expression of LMW cyclin E in HER2+ tumors (p=.04). Conclusions: HER2 downregulation affects post-transcriptional processing of cyclin E resulting in decreased expression of the more tumorigenic LMW forms. LMW forms of cyclin E are overexpressed in a subset of patients with HER2-overexpressing tumors. Further study into the mechanism by which HER2 effects post-transcriptional processing of FL cyclin E to its LMW isoforms may identify a role for LMW cyclin E as a second therapeutic target in patients with HER2 and LMW cyclin E-overexpressing breast cancer.