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HER2 and Cyclin E: Novel Interaction and Opportunities for Therapy
Background: In vitro studies have shown that activation of the HER2 pathway promotes cellular proliferation through shortening of the G1 phase of the cell cycle. One well described regulator of the G1 phase transition is cyclin E. Cyclin E has been identified as a poor prognostic factor in breast cancer patients. Despite the fact that HER2 and cyclin E overexpression are both associated with aggressive forms of breast cancer, a possible relationship between the two has not been investigated. In our preliminary studies, we have used trastuzumab and siRNA to HER2 to downregulate HER2. Both mechanisms resulted in decreased cyclin E expression and activity. Objective/Hypothesis: We hypothesize that HER2 is acting upstream of cyclin E promoting breast tumorigenesis. Our objective is to study the mechanism of ongoing cross-talk between HER2 and cyclin E and to determine if cyclin E plays a role in response to trastuzumab therapy. Specific Aims: (1) Determine the mechanism by which HER2 downregulation decreases cyclin E expression, (2) Investigate the role of HER2 and cyclin E overexpression on response to therapy, (3) Evaluate the prognostic and predictive significance of overexpression of HER2 and cyclin E in breast cancer patients. Study Design: First we will investigate the effects of HER2 on transcription and degradation of cyclin E using HER2 transfected MCF7 cells. The role of the Ras/Raf/MEK/ERK pathway in mediating these effects will be interrogated by inhibiting the pathway at different levels using a Ras dominant negative adenoviral vector and PD098059, a MEK/ERK inhibitor. Second, we will use a model system of MCF-7 cells stably transfected with FLAG-tagged cyclin E constructs (EL1, T1, or T2) representing the full-length (FL) and LMW forms of cyclin E and cotransfected with HER2 in order to investigate the effects of different HER2- and cyclin E-targeted therapies. This model system will allow us to determine the ability of FL and LMW cyclin E levels to predict response to targeted therapy in vitro. Results will be confirmed in vivo using an established HER2-overexpressing breast cancer xenograft model. Finally, we will analyze expression of HER2, FL and LMW cyclin E in breast tissue specimens prospectively collected from patients treated at our institution. HER2, FL and LMW cyclin E levels will be correlated with clinical outcome. Potential Outcomes and Benefits of Research: Our proposed studies will allow us to better understand the mechanism of ongoing cross-talk between HER2 and cyclin E. In addition, insight into the relationship between HER2 and cyclin E, both the FL and LMW forms will determine if cyclin E expression is a potential predictor of response to trastuzumab and otherHER2 specific therapies.
Approximately 30% of breast cancer patients have tumors with an excess amount of the HER2 protein. These patients are likely to have additional poor prognostic factors including disease in their axillary lymph nodes and the absence of estrogen and progesterone receptors in the tumor. These factors contribute to HER2-overexpressing tumors being more aggressive with an increased risk of disease recurrence and death. Trastuzumab is a recombinant humanized monoclonal antibody developed specifically to target HER2 and block its activity. Clinical trials have demonstrated that treatment with trastuzumab alone is an active and well-tolerated option for first-line treatment of HER2-overexpressing metastatic breast cancer. Unfortunately, an objective response is seen in less than 30% of patients. One way that HER2 functions is by shortening the G1 phase of the cell cycle, which normally regulates cell growth. Another protein, cyclin E, has been studied extensively in our laboratory and is known to be an important mediator of the G1 phase transition point. Our preliminary studies show that when HER2-overexpressing breast cancer cells are treated with trastuzumab, levels of both HER2 and cyclin E go down. In addition, if we downregulate HER2 by another method (using siRNA directed at HER2), we again see a decrease in HER2 and cyclin E expression. These data suggest a possible relationship between HER2 and cyclin E, two important prognostic indicators of breast cancer outcome. We propose to study the relationship between HER2 and cyclin E in order to understand how they interact and how we can exploit these interactions for improved therapies for breast cancer patients. First, we will determine the mechanism by which changes in HER2 expression result in changes in cyclin E expression; either increased production through regulation of the gene or through decreased degradation of the cyclin E protein. We will use a model system of breast cancer cells that express the various forms of cyclin E to investigate the effects of different therapies that target HER2. We will also study therapies targeted to cyclin E to determine if interfering with cyclin E activity can improve response to HER2 directed therapies. We will evaluate these interactions between HER2 and cyclin E in cells and in a mouse model of HER2-overexpressing breast cancer. Finally, we will analyze expression of HER2, full-length and low molecular weight cyclin E in breast tissue specimens collected from breast cancer patients. HER2 and full-length and low molecular weight cyclin E levels will be correlated with clinical outcome. Our proposed studies will allow us to better understand the mechanism of cross-talk between HER2 and cyclin E. Insight into the relationship between HER2 and cyclin E will determine if cyclin E expression can predict response to treatment with trastuzumab and other HER2 specific therapies.