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Cyclin E in breast tumors is cleaved into its low molecular weight forms by calpain
Xu Dong Wang1, Jesusa L Rosales, Anthony Magliocco
1Department of Cell Biology and Anatomy, Cancer Biology and Neuroscience Research Groups, The University of Calgary, Alberta, Canada.
Abstract:
Abundant levels of the hyperactive low molecular weight (LMW) forms of cyclin E contribute to deregulation of Cdk2 in breast tumors, but the mechanism through which they arise is not fully understood. Here, we explored the hypothesis that post-translational processing by a protease generates the LMW forms of cyclin E in breast tumors. In ZR75 tumor cell lysates, calcium-induced cyclin E truncation into peptides corresponding in size with LMW forms of cyclin E in tumor tissues. Calpeptin inhibited calcium-stimulated cyclin E truncation, indicating that cleavage resulted from activity of the calcium-dependent protease, calpain. Consistently, calcium+calpain caused truncation of cyclin E immunoprecipitated from tumor cells and tissues. Calcium also caused truncation of the calpain regulatory subunit in tumor cell lysates, indicating that elevated calpain activity accompanies cyclin E truncation. Increased levels of the calpain small subunit were also observed in breast tumors, and significant amounts of its proteolyzed forms indicated increased calpain activity. While elastase also caused cyclin E truncation, the cleavage pattern was distinct from that generated by calpain, suggesting discrete mechanisms in regulating the formation of LMW cyclin E in breast tumors. Treatment of ZR75 cultures with calcium+A23187 recapitulated the formation of the calcium/calpain-induced LMW forms of cyclin E. Altered calcium homeostasis and/or inability of the endogenous calpain inhibitor to control the activity of high levels of the calpain small subunit may contribute to increased calpain activity in breast tumors, causing abundant levels of LMW cyclin E.
Insights
Low molecular weight cyclin E (LMW-cyclin E) in breast tumors is generated by the calcium-dependent protease calpain. This cleavage contributes to Cdk2 deregulation, a key factor in breast cancer progression.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Hyperactive low molecular weight (LMW) forms of cyclin E are abundant in breast tumors.
- These LMW forms contribute to Cdk2 deregulation, a critical event in breast cancer.
- The precise mechanism generating LMW-cyclin E in tumors remains unclear.
Purpose of the Study:
- To investigate the hypothesis that protease-mediated post-translational processing generates LMW-cyclin E in breast tumors.
- To identify the specific protease responsible for cyclin E cleavage in breast cancer.
Main Methods:
- Utilized ZR75 breast tumor cell lysates and tissues.
- Induced cyclin E truncation using calcium and A23187.
- Employed calpeptin to inhibit calcium-stimulated cleavage.
- Performed immunoprecipitation to analyze cyclin E and calpain subunits.
- Compared cleavage patterns generated by calpain and elastase.
Main Results:
- Calcium induced cyclin E truncation in ZR75 cell lysates into peptides consistent with LMW forms found in tumors.
- Calpeptin inhibited this calcium-stimulated truncation, implicating calpain activity.
- Calcium and calpain directly caused cyclin E truncation in immunoprecipitated samples.
- Elevated calpain activity was evidenced by truncation of its regulatory subunit and increased levels of its proteolyzed small subunit in breast tumors.
- Elastase also cleaved cyclin E, but with a distinct pattern, suggesting calpain is the primary enzyme.
Conclusions:
- Calpain, a calcium-dependent protease, is responsible for generating LMW-cyclin E in breast tumors.
- Increased calpain activity, potentially due to altered calcium homeostasis or impaired inhibition, leads to abundant LMW-cyclin E.
- This process contributes to Cdk2 deregulation and may play a significant role in breast cancer development.