Caspase-mediated Apoptosis Mechanisms in Breast Cancer Cells

Institution: The Burnham Institute for Medical Research
Investigator(s): Kelly Boatright, B.S. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8GB-0137 Award: $60,000
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2002)
Genetic instability is an essential feature in the development of all cancers, including those of the breast. Most solid tumors are found to contain various chromosomal abnormalities, some that result from the failure to properly distribute chromosomes between daughter cells during cell division. A failure to properly separate these "sister" chromatids results in a state called aneuploidy. Thus, cancer cells can have extra copies of chromosomes. Our interest is in the process of chromosome separation, the proteins involved, and the identification of the defects associated with genetic instability in cancer. During cell division the sister chromatids are held together by a protein called "ssc1". The cleavage of ssc1 is required to allow proper chromosome segregation and is accomplished by a protease called "separase". In turn, separase activity is controlled by a second protein, called "securin". Our interest is to extend our understanding of this process to breast cancer with the hypothesis that excess securin might be an underlying cause in genetic instability.

The specific aims of this project are to (i) determine the proteolytically active form of separase, (ii) examine the mechanism for modulation of separase activity by securin, and (iii) profile separase activity in breast cancer cells and in normal cells of the breast epithelia and correlate this activity with the levels of securin at the mRNA and protein levels. To accomplish these aims, we propose cloning, over-expression, and biochemical analyses of separase. Cell culture experiments will be done to study separase in HeLa cells. We plan to identify additional proteins that bind to separase using affinity columns.

Cancerous cells lose the ability to halt division at normal cell cycle checkpoints and instead divide unchecked. The resultant abnormal mitoses lead to genetic abnormalities such as aneuploidy, which can provide a further growth advantage and promote the malignant phenotype. A better understanding of how aneuploidy occurs in breast cancer may yield novel ways to prevent the disease.

Final Report (2004)
Note: The PI/mentor changed the focus of the project from the topic of chromosome separation to the new topic of apoptosis.

Overview: The human body contains cells with different life expectancies. Some (white blood cells, skin) are programmed to rapidly die and be replaced. Others (nerve cells) are programmed to survive the lifetime of the individual and are seldom replaced. Our research focuses on the central role enzyme pathways play in the life and death of cells. When death pathways slow down in cells that are normally programmed to die, cancer results. Conversely, when death pathways become overactive in cells that are programmed to survive, degenerative disease occurs. The laboratory focuses on understanding the fundamental molecular interactions that occur within these enzyme pathways. This knowledge is used to engineer synthetic compounds to stimulate cell destruction in cancer cells, or delay cell destruction in neurodegenerative diseases and stroke. (taken from the laboratory summary for Dr. Guy Salvesen, Burnham Institute)

Introduction. These studies have focused on the caspases, a family of proteases that are essential for apoptosis, a form of programmed cell death. Apoptosis is initiated by activation of caspase-8. This activation step is regulated by FLIP, which is a protease-deficient caspase homolog. Once activated, caspase-8 then activates caspases-3 and 7, committing the cell to die. One function of the immune system is to trigger tumor cells to die by apoptosis. This process is known as immunosurveilance. Failure to accomplish this may result in expansion of the tumor population leading to cancer. Therefore, understanding the mechanisms regulating this process is critical to being able to understand why tumor cells fail to die by apoptosis.

Progress. We made progress on a number of aims.
Aim #1- We determined of the mechanism of activation of caspase-8. This work revealed that it is not activated through cleavage, as previously thought. Rather, caspase-8 is activated by dimerization. This challenged conventional dogma of the apoptosis field, and required us to develop new tools to study caspase-8 activation. This work also suggested that FLIP regulation of caspase-8 might not be as clear as previously thought.

Aim #2- We developed the first method for the specific detection of caspase-8 within a biological sample. This work has provided researchers with a tool to study caspase-8 activation, as cleavage alone can no longer be considered a valid assay.

Aim #3- We characterized activation of casinase-8 by FLIP. Based on the findings in Aim #1, I was able to directly test the dimerization hypothesis for caspase-8 activation. We showed that FLIP is indeed an activator of caspase-8, and that it functions through formation of heterodimers with caspase activity.

Impact. The published work from the completed aims of this project reveals important molecular details about the activation of caspase-8. Details of this type are crucial for an understanding of misregulation of immunosurveilance, as well as for the design of therapeutics aimed at eradicating tumors of the breast by initiating caspase-8 activation. By demonstrating a viable method for detecting caspase-8 activation within biological samples, we have provided researchers with tools that can be used to screen for compounds that will induce caspase activation in tumor cell lysates. The studies of activation of caspase-8 by FLIP provides mechanistic insight critical for the rational design of drugs to correct the imbalance between apoptosis and proliferation in breast cancer.

A Unified Model for Apical Caspase Activation
Periodical:Molecular and Cellular Biology
Index Medicus: Mol Cell Biol
Authors: Boatright KM, Renatus M, Scott FL, Sperandio S, Shin H, Petersen IM, Edris, et al.
Yr: 2003 Vol: 11 Nbr: Abs: Pg:529-541

Sequential Autolytic Processing Activates the Zymogen of Arg-Gingipain
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Mikolajczyk J, Boatright KM, Stennicke HR, Nazif T, et al.
Yr: 2002 Vol: 278 Nbr: Abs: Pg:10458-10464

Mechanisms of caspase activation.
Periodical:Current Opinions in Cell Biology
Index Medicus: Curr Opin Cell Biol
Authors: Boatright KM, Salvesen GS.
Yr: 2003 Vol: 15 Nbr: 6 Abs: Pg:725-31

Activation of caspases-8 and -10 by FLIP(L). Biochem J 382.651-657
Periodical:Biochemical Journal
Index Medicus: Biochem J
Authors: Boatright KM, Deis C, Denault JB, Sutherlin DP, Salvesen GS.
Yr: 2004 Vol: 382 Nbr: Abs: Pg:651-7