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Targeting the Cell Cycle in Breast Cancer Treatment
The cell cycle is deregulated in breast cancer and there is overexpression of cyclin E with generation of the low molecular weight isoforms (LMW) due to elastase mediated proteolytic processing. These LMW isoforms have increased cyclin E-CDK2 kinase activity and the detection of the LMW isoforms of cyclin E in human breast cancer is a strong indicator of poor prognosis. We demonstrate that overexpression of E2F1 leads to upregulation of full length cyclin E and generation of the LMW isoforms in nonmalignant breast epithelial cells. This is associated with downregulation of elafin, a known inhibitor of elastase activity. We seek to elucidate the mechanisms by which E2F1 and cyclin E cooperate to deregulate the cell cycle causing generation of the LMW forms of cyclin E. We hypothesize (1) that deregulation of E2F1 in breast epithelial cells leads to the overexpression of cyclin E and processing of the LMW isoforms through downregulation of elafin and (2) that reintroducing elafin in breast cancer cells that express the LMW forms of cyclin E will inhibit growth and lead to apoptosis. We plan to address these hypotheses with three specific aims: (1) To examine the role of E2F1 deregulation in mediating overexpression of cyclin E and downregulation of elafin in breast cell lines, (2) To determine the consequences of E2F1 and elafin overexpression in breast cancer cells in vitro and in vivo, and (3) To correlate E2F1 levels with phosphorylation of pRB and the presence of cyclin E LMW isoforms in primary human breast tumors. These novel interactions between cyclin E and E2F1 have not been previously described and may prove to be important targets in breast cancer treatment. This research has strong translational potential since the LMW forms of cyclin E clearly deregulate the cell cycle in breast cancer, leading to resistance to CDK inhibitors and standard systemic therapies. Understanding the generation and activation of LMW cyclin E will lead to new targeted therapeutics for breast cancer patients.
The cell cycle is characterized by a complete series of events controlled by proteins called cyclins and cyclin dependent kinases (CDKs), which advance the cell cycle, and by CDK inhibitors, which halt the cell cycle. Most cells are not rapidly advancing through the cell cycle and dividing, but rather are in a resting phase. Breast cancer cells are markedly abnormal and their cell cycle is not tightly regulated like normal cells but is significantly deregulated and out of control. One of the cyclins that promotes the cell cycle in breast cancer cells is cyclin E. In normal cells, cyclin E complexes with its partner CDK2 to push the cells out of the resting phase into the proliferating phase. These complexes have a short life lasting only long enough to move cells from one step to another in their cycle. In breast cancer, cyclin E partners with CDK2 in a stronger fashion resulting in a longer lasting complex that pushes cells through the cell cycle over and over again. In addition to the typical cyclin E, breast cancer cells slice up cyclin E into shorter forms that have an even stronger attachment to their CDK2 partner. The shorter forms of cyclin E (termed low molecular weight forms-LMW) are found in 25% of breast cancers and patients whose tumors have LMW forms of cyclin E have a more aggressive form of breast cancer with a greater chance of dying from breast cancer. These shortened forms of cyclin E are a result of an overactive enzyme called elastase. Inhibitors of elastase have been used in clinical studies aimed at reducing inflammation and formation of plaques within blood vessels (atherosclerosis). This proposal shows that exposing normal breast cells to high levels of a protein that regulates cyclin E (E2F1 transcrip-tion factor), causes the LMW forms of cyclin E to appear in normal appearing cells. The E2F1 protein causes LMW forms of cyclin E by decreasing the levels of an inhibitor of elastase (elafin). The elafin protein appears to connect the events of the cell cycle in breast cancer since breast cancer cells have very low levels of elafin but high levels of the shortened forms of cyclin E. We propose to reintroduce elafin into breast cancer cells to reverse the aggressive nature of these cells. By understanding how these proteins control the growth of breast cancer cells, we can develop specific treat-ments aimed directly at inhibiting the shortened forms of cyclin E allowing for improved therapies and a better prognosis for breast cancer patients.